Methods for treating viral infection using il-28 and il-29

ABSTRACT

IL-28A, IL-28B, IL-29, and certain mutants thereof have been shown to have antiviral activity on a spectrum of viral species. Of particular interest is the antiviral activity demonstrated on viruses that infect liver, such as hepatitis B virus and hepatitis C virus. In addition, IL-28A, IL-28B, IL-29, and mutants thereof do not exhibit some of the antiproliferative activity on hematopoietic cells that is observed with interferon treatment. Without the immunosuppressive effects accompanying interferon treatment, IL-28A, IL-28B, and IL-29 will be useful in treating immunocompromised patients for viral infections.

REFERENCE TO RELATED APPLICATIONS

The present application is a divisional of U.S. patent application Ser.No. 10/691,923, filed Oct. 23, 2003, which claims the benefit of U.S.Provisional Application Ser. No. 60/420,714, filed Oct. 23, 2002, U.S.Provisional Application Ser. No. 60/463,939, filed Apr. 18, 2003, U.S.Provisional Application Ser. No. 60/420,713, filed Oct. 23, 2002, andU.S. Provisional Application Ser. No. 60/463,982, filed Apr. 18, 2003,all of which are herein incorporated by reference.

BACKGROUND OF THE INVENTION

Strategies for treating infectious disease often focus on ways toenhance immunity. For instance, the most common method for treatingviral infection involves prophylactic vaccines that induce immune-basedmemory responses. Another method for treating viral infection includespassive immunization via immunoglobulin therapy (Meissner, J. Pediatr.124:S17-21, 1994). Administration of Interferon alpha (IFN-α) is anothermethod for treating viral infections such as genital warts (Reichman etal., Ann. Intern. Med. 108:675-9, 1988) and chronic viral infectionslike hepatitis C virus (HCV) (Davis et al., New Engl. J. Med.339:1493-9, 1998) and hepatitis B virus (HBV). For instance, IFN-α andIFN-β are critical for inhibiting virus replication (reviewed by Vileeket al., (Eds.), Interferons and other cytokines. In Fields FundamentalVirology., 3^(rd) ed., Lippincott-Raven Publishers Philadelphia, Pa.,1996, pages 341-365). In response to viral infection, CD4+ T cellsbecome activated and initiate a T-helper type I (TH1) response and thesubsequent cascade required for cell-mediated immunity. That is,following their expansion by specific growth factors like the cytokineIL-2, T-helper cells stimulate antigen-specific CD8+ T-cells,macrophages, and NK cells to kill virally infected host cells. Althoughoftentimes efficacious, these methods have limitations in clinical use.For instance, many viral infections are not amenable to vaccinedevelopment, nor are they treatable with antibodies alone. In addition,IFN's are not extremely effective and they can cause significanttoxicities; thus, there is a need for improved therapies.

Not all viruses and viral diseases are treated identically becausefactors, such as whether an infection is acute or chronic and thepatient's underlying health, influence the type of treatment that isrecommended. Generally, treatment of acute infections in immunocompetentpatients should reduce the disease's severity, decrease complications,and decrease the rate of transmission. Safety, cost, and convenience areessential considerations in recommending an acute antiviral agent.Treatments for chronic infections should prevent viral damage to organssuch as liver, lungs, heart, central nervous system, andgastrointestinal sytem, making efficacy the primary consideration.

Chronic hepatitis is one of the most common and severe viral infectionsof humans worldwide belonging to the Hepadnaviridae family of viruses.Infected individuals are at high risk for developing liver cirrhosis,and eventually, hepatic cancer. Chronic hepatitis is characterized as aninflammatory liver disease continuing for at least six months withoutimprovement. The majority of patients suffering from chronic hepatitisare infected with either chronic HBV, HCV or are suffering fromautoimmune disease. The prevalence of HCV infection in the generalpopulation exceeds 1% in the United States, Japan, China and SoutheastAsia.

Chronic HCV can progress to cirrhosis and extensive necrosis of theliver. Although chronic HCV is often associated with deposition of typeI collagen leading to hepatic fibrosis, the mechanisms of fibrogenesisremain unknown. Liver (hepatic) fibrosis occurs as a part of thewound-healing response to chronic liver injury. Fibrosis occurs as acomplication of haemochromatosis, Wilson's disease, alcoholism,schistosomiasis, viral hepatitis, bile duct obstruction, toxin exposure,and metabolic disorders. This formation of scar tissue is believed torepresent an a t tempt by the body to encapsulate the injured tissue.Liver fibrosis is characterized by the accumulation of extracellularmatrix that can be distinguished qualitatively from that in normalliver. Left unchecked, hepatic fibrosis progresses to cirrhosis (definedby the presence of encapsulated nodules), liver failure, and death.

There are few effective treatments for hepatitis. For example, treatmentof autoimmune chronic hepatitis is generally limited toimmunosuppressive treatment with corticosteroids. For the treatment ofHBV and HCV, the FDA has approved administration of recombinant IFN-α.However, IFN-α is associated with a number of dose-dependent adverseeffects, including thrombocytopenia, leukopenia, bacterial infections,and influenza-like symptoms. Other agents used to treat chronic HBV orHCV include the nucleoside analog RIBAVIRIN™ and ursodeoxycholic acid;however, neither has been shown to be very effective. RIBAVIRIN™+IFNcombination therapy for results in 47% rate of sustained viral clearance(Lanford, R. E. and Bigger, C. Virology 293:1-9 (2002). (See Medicine,(D. C. Dale and D. D. Federman, eds.) (Scientific American, Inc., NewYork), 4:VIII:1-8 (1995)).

Respiratory syncytial virus is the major cause of pneumonia andbronchiolitis in infancy. RSV infects more than half of infants duringtheir first year of exposure, and nearly all are infected after a secondyear. During seasonal epidemics most infants, children, and adults areat risk for infection or reinfection. Other groups at risk for seriousRSV infections include premature infants, immune compromised childrenand adults, and the elderly. Symptoms of RSV infection range from a mildcold to severe bronchiolitis and pneumonia. Respiratory syncytial virushas also been associated with acute otitis media and RSV can berecovered from middle ear fluid. Herpes simplex virus-1 (HSV-1) andherpes simplex virus-2 (HSV-2) may be either lytic or latent, and arethe causative agents in cold sores (HSV-1) and genital herpes, typicallyassociated with lesions in the region of the eyes, mouth, and genitals(HSV-2). These viruses are a few examples of the many viruses thatinfect humans for which there are few adequate treatments available onceinfection has occurred.

The demonstrated activities of the IL-28 and IL-29 cytokine familyprovides methods for treating specific viral infections, in particular,liver specific viral infections. The activity of IL-28 and IL-29 alsodemonstrate that these cytokines provide methods for treatingimmunocompromised patients. The methods for these and other uses thatshould be apparent to those skilled in the art from the teachingsherein.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides methods for treating viralinfections comprising administering to a mammal with a viral infectioncausing liver inflammation a therapeutically effective amount of apolypeptide comprising an amino acid sequence that has at least 95%identity to SEQ ID NO:2 from amino acid residue 22 to residue 205,wherein after administration of the polypeptide the viral infectionlevel or liver inflammation is reduced. In other embodiments, themethods comprise administering polypeptide comprising an amino acidsequence as shown in SEQ ID NO:18, SEQ ID NO:24, SEQ ID NO:26, SEQ IDNO:28, SEQ ID NO:30, or SEQ ID NO:36. In other embodiments, thepolypeptide is conjugated to a polyalkyl oxide moiety, such as PEG. Inanother embodiment, a reduction in the viral infection level is measuredas a decrease in viral load, an increase in antiviral antibodies, adecrease in serological levels of alanine aminotransferase orhistological improvement. In another embodiment, the mammal is a human.In another embodiment, the present invention provides that the viralinfection is a hepatitis B virus infection or a hepatitis C virusinfection.

In another aspect, the present invention provides methods of treating aviral infection comprising administering to a mammal with a viralinfection causing liver inflammation a therapeutically effective amountof polypeptide comprising an amino acid sequence that has at least 95%identity to SEQ ID NO:4 from amino acid residue 20 to residue 200,wherein after administration of the polypeptide the viral infection isreduced. In another embodiments, the polypeptide comprises an amino acidsequence as shown in SEQ ID NO:20, SEQ ID NO:32, SEQ ID NO:34, or SEQ IDNO:38. In another embodiment, the polypeptide is conjugated to apolyalkyl oxide moiety, such as PEG. In another embodiment, thereduction in the viral infection levels is measured as a decrease inviral load, an increase in antiviral antibodies, a decrease inserological levels of alanine aminotransferase or histologicalimprovement. In another embodiment, the mammal is a human. In anotherembodiment, the viral infection is a hepatitis B virus infection or ahepatitis C virus infection.

In another aspect, the present invention provides methods for treatingliver inflammation comprising administering to a mammal in need thereofa therapeutically effective amount of a polypeptide comprising an aminoacid sequence that has at least 95% identity to SEQ ID NO:2 from aminoacid residue 22 to residue 205, wherein after administration of thepolypeptide the liver inflammation is reduced. In one embodiment, theinvention provides that the polypeptide comprises an amino acid sequenceas shown in SEQ ID NO:18, SEQ ID NO:28, SEQ ID NO:30; SEQ ID NO:24, SEQID NO:26 or SEQ ID NO:36. In another embodiment, the polypeptide isconjugated to a polyalkyl oxide moiety, such as PEG. In anotherembodiment, the present invention provides that the reduction in theliver inflammation is measured as a decrease in serological level ofalanine aminotransferase or histological improvement. In anotherembodiment, the mammal is a human. In another embodiment, the liverinflammation is associated with a hepatitis C virus infection or ahepatitis B virus infection.

In another aspect, the present invention provides methods of treating aviral infection comprising administering to an immunocompromised mammalwith an viral infection a therapeutically effective amount of apolypeptide comprising an amino acid sequence that has at least 95%identity to SEQ ID NO:2 from amino acid residue 22 to amino acid residue205, wherein after administration of the polypeptide the viral infectionis reduced. In another embodiment, the polypeptide comprises an aminoacid sequence as shown in SEQ ID NO:18, SEQ ID NO:28, SEQ ID NO:30; SEQID NO:24, SEQ ID NO:26 or SEQ ID NO:36. In another embodiment, thepolypeptide is conjugated to a polyalkyl oxide moiety, such as PEG. Inanother embodiment, a reduction in the viral infection level is measuredas a decrease in viral load, an increase in antiviral antibodies, adecrease in serological levels of alanine aminotransferase orhistological improvement. In another embodiment, the mammal is a human.In another embodiment, the present invention provides that the viralinfection is a hepatitis B virus infection or a hepatitis C virusinfection.

In another aspect, the present invention provides methods of treating aviral infection comprising administering to an immunocompromised mammalwith a viral infection a therapeutically effective amount of apolypeptide comprising an amino acid sequence that has at least 95%identity to SEQ ID NO:4 from amino acid residue 20 to residue 200,wherein after administration of the polypeptide the viral infection isreduced. In other embodiments, the polypeptide comprises an amino acidsequence as shown in SEQ ID NO:20, SEQ ID NO:32, SEQ ID NO:34, or SEQ IDNO:38. In another embodiment, the polypeptide is conjugated to apolyalkyl oxide moiety, such as PEG. In another embodiment, thereduction in the viral infection levels is measured as a decrease inviral load, an increase in antiviral antibodies, a decrease inserological levels of alanine aminotransferase or histologicalimprovement. In another embodiment, the mammal is a human. In anotherembodiment, the viral infection is a hepatitis B virus infection or ahepatitis C virus infection.

In another aspect, the present invention provides methods of treatingliver inflammation comprising administering to an immunocompromisedmammal with liver inflammation a therapeutically effective amount of apolypeptide comprising an amino acid sequence that has at least 95%identity to SEQ ID NO:2 from amino acid residue 22 to amino acid residue205, wherein after administration of the polypeptide the liverinflammation is reduced. In another embodiment, the polypeptidecomprises an amino acid sequence as shown in SEQ ID NO:18, SEQ ID NO:28,SEQ ID NO:30; SEQ ID NO:24, SEQ ID NO:26 or SEQ ID NO:36. In anotherembodiment, the polypeptide is conjugated to a polyalkyl oxide moiety,such as PEG. In another embodiment, a reduction in the liverinflammation level is measured as a decrease in serological levels ofalanine aminotransferase or histological improvement. In anotherembodiment, the mammal is a human. In another embodiment, the presentinvention provides that the viral infection is a hepatitis B virusinfection or a hepatitis C virus infection.

In another aspect, the present invention provides methods of treatingliver inflammation comprising administering to an immunocompromisedmammal with liver inflammation a therapeutically effective amount of apolypeptide comprising an amino acid sequence that has at least 95%identity to SEQ ID NO:4 from amino acid residue 20 to residue 200,wherein after administration of the polypeptide the liver inflammationis reduced. In other embodiments, the polypeptide comprises an aminoacid sequence as shown in SEQ ID NO:20, SEQ ID NO:32, SEQ ID NO:34, orSEQ ID NO:38. In another embodiment, the polypeptide is conjugated to apolyalkyl oxide moiety, such as PEG. In another embodiment, thereduction in the liver inflammation is measured as a decrease inserological levels of alanine aminotransferase or histologicalimprovement. In another embodiment, the mammal is a human. In anotherembodiment, the viral infection is a hepatitis B virus infection or ahepatitis C virus infection.

DESCRIPTION OF THE INVENTION

Prior to setting forth the invention in detail, it may be helpful to theunderstanding thereof to define the following terms:

The term “affinity tag” is used herein to denote a polypeptide segmentthat can be attached to a second polypeptide to provide for purificationor detection of the second polypeptide or provide sites for attachmentof the second polypeptide to a substrate. In principal, any peptide orprotein for which an antibody or other specific binding agent isavailable can be used as an affinity tag. Affinity tags include apoly-histidine tract, protein A (Nilsson et al., EMBO J. 4:1075, 1985;Nilsson et al., Methods Enzymol. 198:3, 1991), glutathione S transferase(Smith and Johnson, Gene 67:31, 1988), Glu-Glu affinity tag(Grussenmeyer et al., Proc. Natl. Acad. Sci. USA 82:7952-4, 1985),substance P, Flag™ peptide (Hopp et al., Biotechnology 6:1204-10, 1988),streptavidin binding peptide, or other antigenic epitope or bindingdomain. See, in general, Ford et al., Protein Expression andPurification 2:95-107, 1991. DNAs encoding affinity tags are availablefrom commercial suppliers (e.g., Pharmacia Biotech, Piscataway, N.J.).

The term “allelic variant” is used herein to denote any of two or morealternative forms of a gene occupying the same chromosomal locus.Allelic variation arises naturally through mutation, and may result inphenotypic polymorphism within populations. Gene mutations can be silent(no change in the encoded polypeptide) or may encode polypeptides havingaltered amino acid sequence. The term allelic variant is also usedherein to denote a protein encoded by an allelic variant of a gene.

The terms “amino-terminal” and “carboxyl-terminal” are used herein todenote positions within polypeptides. Where the context allows, theseterms are used with reference to a particular sequence or portion of apolypeptide to denote proximity or relative position. For example, acertain sequence positioned carboxyl-terminal to a reference sequencewithin a polypeptide is located proximal to the carboxyl terminus of thereference sequence, but is not necessarily at the carboxyl terminus ofthe complete polypeptide.

The term “complement/anti-complement pair” denotes non-identicalmoieties that form a non-covalently associated, stable pair underappropriate conditions. For instance, biotin and avidin (orstreptavidin) are prototypical members of a complement/anti-complementpair. Other exemplary complement/anti-complement pairs includereceptor/ligand pairs, antibody/antigen (or hapten or epitope) pairs,sense/antisense polynucleotide pairs, and the like. Where subsequentdissociation of the complement/anti-complement pair is desirable, thecomplement/anti-complement pair preferably has a binding affinity of<10⁹ M⁻¹.

The term “degenerate nucleotide sequence” denotes a sequence ofnucleotides that includes one or more degenerate codons (as compared toa reference polynucleotide molecule that encodes a polypeptide).Degenerate codons contain different triplets of nucleotides, but encodethe same amino acid residue (i.e., GAU and GAC triplets each encodeAsp).

The term “expression vector” is used to denote a DNA molecule, linear orcircular, that comprises a segment encoding a polypeptide of interestoperably linked to additional segments that provide for itstranscription. Such additional segments include promoter and terminatorsequences, and may also include one or more origins of replication, oneor more selectable markers, an enhancer, a polyadenylation signal, etc.Expression vectors are generally derived from plasmid or viral DNA, ormay contain elements of both.

The term “isolated”, when applied to a polynucleotide, denotes that thepolynucleotide has been removed from its natural genetic milieu and isthus free of other extraneous or unwanted coding sequences, and is in aform suitable for use within genetically engineered protein productionsystems. Such isolated molecules are those that are separated from theirnatural environment and include cDNA and genomic clones. Isolated DNAmolecules of the present invention are free of other genes with whichthey are ordinarily associated, but may include naturally occurring 5′and 3′ untranslated regions such as promoters and terminators. Theidentification of associated regions will be evident to one of ordinaryskill in the art (see for example, Dynan and Tijan, Nature 316:774-78,1985).

An “isolated” polypeptide or protein is a polypeptide or protein that isfound in a condition other than its native environment, such as apartfrom blood and animal tissue. In a preferred form, the isolatedpolypeptide is substantially free of other polypeptides, particularlyother polypeptides of animal origin. It is preferred to provide thepolypeptides in a highly purified form, i.e. greater than 95% pure, morepreferably greater than 99% pure. When used in this context, the term“isolated” does not exclude the presence of the same polypeptide inalternative physical forms, such as dimers or alternatively glycosylatedor derivatized forms.

The term “level” when referring to immune cells, such as NK cells, Tcells, in particular cytotoxic T cells, B cells and the like, anincreased level is either increased number of cells or enhanced activityof cell function.

The term “level” when referring to viral infections refers to a changein the level of viral infection and includes, but is not limited to, achange in the level of CTLs or NK cells (as described above), a decreasein viral load, an increase antiviral antibody titer, decrease inserological levels of alanine aminotransferase, or improvement asdetermined by histological examination of a target tissue or organ.Determination of whether these changes in level are significantdifferences or changes is well within the skill of one in the art.

The term “operably linked”, when referring to DNA segments, indicatesthat the segments are arranged so that they function in concert fortheir intended purposes, e.g., transcription initiates in the promoterand proceeds through the coding segment to the terminator.

The term “ortholog” denotes a polypeptide or protein obtained from onespecies that is the functional counterpart of a polypeptide or proteinfrom a different species. Sequence differences among orthologs are theresult of speciation.

“Paralogs” are distinct but structurally related proteins made by anorganism. Paralogs are believed to arise through gene duplication. Forexample, a α-globin, β-globin, and myoglobin are paralogs of each other.

A “polynucleotide” is a single- or double-stranded polymer ofdeoxyribonucleotide or ribonucleotide bases read from the 5′ to the 3′end. Polynucleotides include RNA and DNA, and may be isolated fromnatural sources, synthesized in vitro, or prepared from a combination ofnatural and synthetic molecules. Sizes of polynucleotides are expressedas base pairs (abbreviated “bp”), nucleotides (“nt”), or kilobases(“kb”). Where the context allows, the latter two terms may describepolynucleotides that are single-stranded or double-stranded. When theterm is applied to double-stranded molecules it is used to denoteoverall length and will be understood to be equivalent to the term “basepairs”. It will be recognized by those skilled in the art that the twostrands of a double-stranded polynucleotide may differ slightly inlength and that the ends thereof may be staggered as a result ofenzymatic cleavage; thus all nucleotides within a double-strandedpolynucleotide molecule may not be paired.

A “polypeptide” is a polymer of amino acid residues joined by peptidebonds, whether produced naturally or synthetically. Polypeptides of lessthan about 10 amino acid residues are commonly referred to as“peptides”.

The term “promoter” is used herein for its art-recognized meaning todenote a portion of a gene containing DNA sequences that provide for thebinding of RNA polymerase and initiation of transcription. Promotersequences are commonly, but not always, found in the 5′ non-codingregions of genes.

A “protein” is a macromolecule comprising one or more polypeptidechains. A protein may also comprise non-peptidic components, such ascarbohydrate groups. Carbohydrates and other non-peptidic substituentsmay be added to a protein by the cell in which the protein is produced,and will vary with the type of cell. Proteins are defined herein interms of their amino acid backbone structures; substituents such ascarbohydrate groups are generally not specified, but may be presentnonetheless.

The term “receptor” denotes a cell-associated protein that binds to abioactive molecule (i.e., a ligand) and mediates the effect of theligand on the cell. Membrane-bound receptors are characterized by amulti-peptide structure comprising an extracellular ligand-bindingdomain and an intracellular effector domain that is typically involvedin signal transduction. Binding of ligand to receptor results in aconformational change in the receptor that causes an interaction betweenthe effector domain and other molecule(s) in the cell. This interactionin turn leads to an alteration in the metabolism of the cell. Metabolicevents that are linked to receptor-ligand interactions include genetranscription, phosphorylation, dephosphorylation, increases in cyclicAMP production, mobilization of cellular calcium, mobilization ofmembrane lipids, cell adhesion, hydrolysis of inositol lipids andhydrolysis of phospholipids. In general, receptors can be membranebound, cytosolic or nuclear; monomeric (e.g., thyroid stimulatinghormone receptor, beta-adrenergic receptor) or multimeric (e.g., PDGFreceptor, growth hormone receptor, IL-3 receptor, GM-CSF receptor, G-CSFreceptor, erythropoietin receptor and IL-6 receptor).

The term “secretory signal sequence” denotes a DNA sequence that encodesa polypeptide (a “secretory peptide”) that, as a component of a largerpolypeptide, directs the larger polypeptide through a secretory pathwayof a cell in which it is synthesized. The larger polypeptide is commonlycleaved to remove the secretory peptide during transit through thesecretory pathway.

The term “splice variant” is used herein to denote alternative forms ofRNA transcribed from a gene. Splice variation arises naturally throughuse of alternative splicing sites within a transcribed RNA molecule, orless commonly between separately transcribed RNA molecules, and mayresult in several mRNAs transcribed from the same gene. Splice variantsmay encode polypeptides having altered amino acid sequence. The termsplice variant is also used herein to denote a protein encoded by asplice variant of an mRNA transcribed from a gene.

Molecular weights and lengths of polymers determined by impreciseanalytical methods (e.g., gel electrophoresis) will be understood to beapproximate values. When such a value is expressed as “about” X or“approximately” X, the stated value of X will be understood to beaccurate to ±10%.

“zcyto20”, “zcyto21”, “zcyto22” are the previous designations for humanIL-28A, IL-29, and IL-28B. IL-28A nucleotide and amino acid sequencesare shown in SEQ ID NOS: 1, 2, 17, 18, 35, 36. IL-28B nucleotide andamino acid sequences are shown in SEQ ID NOS: 5, 6,21, 22, 39, 40. IL-29nucleotide and amino acid sequences are shown in SEQ ID NOS: 3, 4, 19,20, 37, 38. These sequences are described in PCT application WO02/086087 and U.S. Provisional Patent Application No. 60/493,194, bothcommonly assigned to ZymoGenetics, Inc., incorporated herein byreference.

“zcyto24” and “zcyto25” are the previous designations for mouse IL-28Aand IL-28B, and are shown in SEQ ID NOS: 7, 8, 9, 10, respectively. Thepolynucleotide and polypeptides are fully described in PCT applicationWO 02/086087 commonly assigned to ZymoGenetics, Inc., incorporatedherein by reference.

“zcytor19” is the previous designation for IL-28 receptor α-subunit, andis shown in SEQ ID NOS: 11, 12, 13, 14, 15, 16. The polynucleotides andpolypeptides are described in PCT application WO 02/20569 on behalf ofSchering, Inc., and WO 02/44209 assigned to ZymoGenetics, Inc andincorporated herein by reference. “IL-28 receptor” denotes the IL-28α-subunit and CRF2-4 subunit forming a heterodimeric receptor.

All references cited herein are incorporated by reference in theirentirety.

We have previously reported the discovery of a new family ofinterferon-like molecules in PCT applications, PCT/US01/21087 andPCT/US02/12887, and Sheppard et al., Nature Immunol. 4:63-68, 2003;herein all incorporated by reference. This new family includes moleculesdesignated zcyto20 (SEQ ID NOS: 1 and 2; 17 and 18), zcyto21 (SEQ IDNOS:3 and 4; 19 and 20), zcyto22 (SEQ ID NOS:5 and 6; 21 and 22),zcyto24 (SEQ ID NOS:7 and 8), zcyto25 (SEQ ID NOS: 9 and 10), wherezcyto20, 21, and 22 are human sequences, and zcyto24 and 25 are mousesequences. HUGO designations have been assigned to the interferon-likeproteins. Zcyto20 has been designated IL-28A, zycto22 has beendesignated IL-28B, zycto21 has been designated IL-29, and will bereferred to by the HUGO names hereafter. Kotenko et al., Nature Immunol.4:69-77, 2003, have identified IL-28A as IFNλ2, IL-28B as IFNλ3, andIL-29 as IFNλ1. The receptor for these proteins, originally designatedzcytor19 (SEQ ID NOS: 11 and 12), has been designated as IL-28RA byHUGO.

The present invention provides methods for using IL-28 and IL-29 as anantiviral agent in a broad spectrum of viral infections. In certainembodiments, the methods include using IL-28 and IL-29 in viralinfections that are specific for liver, such as hepatitis. Furthermore,data indicate that IL-28 and IL-29 exhibit these antiviral activitieswithout some of the toxicities associated with the use of IFN therapyfor viral infection. One of the toxicities related to type I interferontherapy is myelosuppression. This is due to type I interferonssuppression of bone marrow progenitor cells. Because IL-29 does notsignificantly suppress bone marrow cell expansion or B cellproliferation as is seen with IFN-α, IL-29 will have less toxicityassociated with treatment. Similar results would be expected with IL-28Aand IL-28B.

IFN-α may be contraindicated in some patients, particularly when dosessufficient for efficacy have some toxicity or myelosuppressive effects.Examples of patients for which IFN is contraindicated can include (1)patients given previous immunosuppressive medication, (2) patients withHIV or hemophilia, (3) patients who are pregnant, (4) patients with acytopenia, such as leukocyte deficiency, neutropenia, thrombocytopenia,and (5) patients exhibiting increased levels of serum liver enzymes.Moreover, IFN therapy is associated with symptoms that are characterizedby nausea, vomiting, diarrhea and anorexia. The result being that somepopulations of patients will not tolerate IFN therapy, and IL-28A,IL-28B, and IL-29 can provide an alternative therapy for some of thosepatients.

The methods of the present invention comprise administering atherapeutically effective amount of IL-28A, IL-28B, IL-29, or mutant ofsaid molecules that have retained some biological activity associatedwith IL-28A, IL-28B or IL-29, alone or in combination with otherbiologics or pharmaceuticals. The present invention provides methods oftreating a mammal with a chronic or acute viral infection, causing liverinflammation, thereby reducing the viral infection or liverinflammation. In another aspect, the present invention provides methodsof treating liver specific diseases, in particular liver disease whereviral infection is in part an etiologic agent. These methods are basedon the discovery that IL-28 and IL-29 have antiviral activity on hepaticcells.

As stated above, the methods of the present invention provideadministering a therapeutically effective amount of IL-28A, IL-28B,IL-29, or mutant of said molecules that have retained some biologicalactivity associated with IL-28A, IL-28B or IL-29, alone or incombination with other biologics or pharmaceuticals. The presentinvention provides methods of treatment of a mammal with a viralinfection selected from the group consisting of hepatitis A, hepatitisB, hepatitis C, and hepatitis D. Other aspects of the present inventionprovide methods for using IL-28 or IL-29 as an antiviral agent in viralinfections selected from the group consisting of respiratory syncytialvirus, herpes virus, Epstein-Barr virus, influenza virus, adenovirus,parainfluenza virus, rhino virus, coxsackie virus, vaccinia virus, westnile virus, dengue virus, Venezuelan equine encephalitis virus, pichindevirus and polio virus. In certain embodiments, the mammal can haveeither a chronic or acute viral infection.

In another aspect, the methods of the present invention also include amethod of treating a viral infection comprising administering atherapeutically effective amount of IL-28A, IL-28B, IL-29, or mutant ofsaid molecules that have retained some biological activity associatedwith IL-28A, IL-28B or IL-29, alone or in combination with otherbiologics or pharmaceuticals, to an immunompromised mammal with a viralinfection, thereby reducing the viral infection, such as is describedabove. All of the above methods of the present invention can alsocomprise the administration of zcyto24 or zcyto25 as well.

Description of IL-28A, IL-28B, and IL-29 Polynucleotides andPolypeptides

An IL-28A gene encodes a polypeptide of 205 amino acids, as shown in SEQID NO:2. The signal sequence for IL-28A comprises amino acid residue 1(Met) through amino acid residue 21 (Ala) of SEQ ID NO:2. The maturepeptide for IL-28A begins at amino acid residue 22 (Val). A variantIL-28A gene encodes a polypeptide of 200 amino acids, as shown in SEQ IDNO:18. The signal sequence for IL-28A can be predicted as comprisingamino acid residue-25 (Met) through amino acid residue-1 (Ala) of SEQ IDNO:18. The mature peptide for IL-28A begins at amino acid residue 1(Val). IL-28A helices are predicted as follow: helix A is defined byamino acid residues 24 (Leu) to 40 (Glu); helix B by amino acid residues58 (Thr) to 65 (Gln); helix C by amino acid residues 69 (Arg) to 85(Ala); helix D by amino acid residues 95 (Val) to 114 (Ala); helix E byamino acid residues 126 (Thr) to 142 (Lys); and helix F by amino acidresidues 148 (Cys) to 169 (Ala); as shown in SEQ ID NO: 18. When apolynucleotide sequence encoding the mature polypeptide is expressed ina prokaryotic system, such as E. coli , the a secretory signal sequencemay not be required and the an N-terminal Met will be present, resultingin expression of a polypeptide such as is shown in SEQ ID NO:36.

The IL-29 gene encodes a polypeptide of 200 amino acids, as shown in SEQID NO:4. The signal sequence for IL-29 comprises amino acid residue 1(Met) through amino acid residue 19 (Ala) of SEQ ID NO:4. The maturepeptide for IL-29 begins at amino acid residue 20 (Gly). IL-29 has beendescribed in published PCT application WO 02/02627. A variant IL-29 geneencodes a polypeptide of 200 amino acids, as shown in SEQ ID NO:20,where amino acid residue 169 is Asn instead of Asp. The signal sequencefor IL-29 can be predicted as comprising amino acid residue-19 (Met)through amino acid residue-1 (Ala) of SEQ ID NO:20. The mature peptidefor IL-29 begins at amino acid residue 1 (Gly). IL-29 has been describedin PCT application WO 02/02627. IL-29 helices are predicted as follows:helix A is defined by amino acid residues 30 (Ser) to 44 (Leu); helix Bby amino acid residues 57 (Asn) to 65 (Val); helix C by amino acidresidues 70 (Val) to 85 (Ala); helix D by amino acid residues 92 (Glu)to 114 (Gln); helix E by amino acid residues 118 (Thr) to 139 (Lys); andhelix F by amino acid residues 144 (Gly) to 170 (Leu); as shown in SEQID NO: 20. When a polynucleotide sequence encoding the maturepolypeptide is expressed in a prokaryotic system, such as E. coli , thea secretory signal sequence may not be required and the an N-terminalMet will be present, resulting in expression of a polypeptide such as isshown in SEQ ID NO:38.

The IL-28B gene encodes a polypeptide of 205 amino acids, as shown inSEQ ID NO:6. The signal sequence for IL-28B comprises amino acid residue1 (Met) through amino acid residue 21 (Ala) of SEQ ID NO:6. The maturepeptide for IL-28B begins at amino acid residue 22 (Val). A variantIL-28B gene encodes a polypeptide of 200 amino acids, as shown in SEQ IDNO:22. The signal sequence for IL-28B can be predicted as comprisingamino acid residue-21 (Met) through amino acid residue-1 (Ala) of SEQ IDNO:22. The mature peptide for IL-28B begins at amino acid residue 1(Val). IL-28B helices are predicted as follow: helix A is defined byamino acid residues 8 (Leu) to 41 (Glu); helix B by amino acid residues58 (Trp) to 65 (Gln); helix C by amino acid residues 69 (Arg) to 86(Ala); helix D by amino acid residues 95 (Gly) to 114 (Ala); helix E byamino acid residues 126 (Thr) to 142 (Lys); and helix F by amino acidresidues 148 (Cys) to 169 (Ala); as shown in SEQ ID NO: 2. When apolynucleotide sequence encoding the mature polypeptide is expressed ina prokaryotic system, such as E. coli , the a secretory signal sequencemay not be required and the an N-terminal Met will be present, resultingin expression of a polypeptide such as is shown in SEQ ID NO:40.

Zcyto24 gene encodes a polypeptide of 202 amino acids, as shown in SEQID NO:8. Zcyto24 secretory signal sequence comprises amino acid residue1 (Met) through amino acid residue 28 (Ala) of SEQ ID NO:8. Analternative site for cleavage of the secretory signal sequence can befound at amino acid residue 24 (Thr). The mature polypeptide comprisesamino acid residue 29 (Asp) to amino acid residue 202 (Val).

Zcyto25 gene encodes a polypeptide of 202 amino acids, as shown in SEQID NO:10. Zcyto25 secretory signal sequence comprises amino acid residue1 (Met) through amino acid residue 28 (Ala) of SEQ ID NO:10. Analternative site for cleavage of the secretory signal sequence can befound at amino acid residue 24 (Thr). The mature polypeptide comprisesamino acid residue 29 (Asp) to amino acid residue 202 (Val).

The present invention provides methods comprising administration ofpolypeptides having mutations in the IL-28 and IL-29 wildtype sequencethat result in expression of single forms of the IL-28 or IL-29molecule. Exemplary mutants are shown in SEQ ID NOS: 24, 28, and 32.When IL-28 and IL-29 are expressed in E. coli, an N-terminal Methionineis present. SEQ ID NOS: 26, 30 and 34 show the amino acid residuenumbering for IL-28A and IL-29 mutants when the N-terminal Met ispresent. Table 1 shows the possible combinations of intramoleculardisulfide bonded cysteine pairs for wildtype I L-28A, IL-28B, and IL-29.TABLE 1 IL-28A C₁₆-C₁₁₅ C₄₈-C₁₄₈ C₅₀-C₁₄₈ C₁₆₇-C₁₇₄ C₁₆-C₄₈ C₁₆-C₅₀C₄₈-C₁₁₅ C₅₀-C₁₁₅ C₁₁₅-C₁₄₈ SEQ ID NO: 18 Met IL- C₁₇-C₁₁₆ C₄₉-C₁₄₉C₅₁-C₁₄₉₈ C₁₆₈-C₁₇₅ C₁₇-C₄₉ C₁₇-C₅₁ C₄₉-C₁₁₆ C₅₁-C₁₁₆ C₁₁₆-C₁₄₉ 28A SEQID NO: 36 IL-29 C₁₅-C₁₁₂ C₄₉-C₁₄₅ C₁₁₂-C₁₇₁ SEQ ID NO: 20 Met IL-29C₁₆-C₁₁₃ C₅₀-C₁₄₆ C₁₁₃-C₁₇₂ SEQ ID NO: 38 IL-28B C₁₆-C₁₁₅ C₄₈-C₁₄₈C₅₀-C₁₄₈ C₁₆₇-C₁₇₄ C₁₆-C₄₈ C₁₆-C₅₀ C₄₈-C₁₁₅ C₅₀-C₁₁₅ C₁₁₅-C₁₄₈ SEQ IDNO: 22 Met IL-28B C₁₇-C₁₁₆ C₄₉-C₁₄₉ C₅₁-C₁₄₉₈ C₁₆₈-C₁₇₅ C₁₇-C₄₉ C₁₇-C₅₁C₄₉-C₁₁₆ C₅₁-C₁₁₆ C₁₁₆-C₁₄₉ SEQ ID NO: 40

Conjugation of interferons with water-soluble polymers has been shown toenhance the circulating half-life of the interferon, and to reduce theimmunogenicity of the polypeptide (see, for example, Nieforth et al,Clin. Pharmacol. Ther. 59:636 (1996), and Monkarsh et al, Anal. Biochem.247:434 (1997)). Suitable water-soluble polymers include polyethyleneglycol (PEG), monomethoxy-PEG, mono-(C1-C10)alkoxy-PEG, aryloxy-PEG,poly-(N-vinyl pyrrolidone)PEG, tresyl monomethoxy PEG, PEGpropionaldehyde, bis-succinimidyl carbonate PEG, propylene glycolhomopolymers, a polypropylene oxide/ethylene oxide co-polymer,polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, dextran,cellulose, or other carbohydrate-based polymers. Suitable PEG may have amolecular weight from about 600 to about 60,000, including, for example,5,000, 12,000, 20,000 and 40,000. A Cysteine mutant IL-28 or IL-29conjugate can also comprise a mixture of such water-soluble polymers.

One example of a IL-28 or IL-29 conjugate comprises a IL-28 or IL-29moiety, or mutant thereof, and a polyalkyl oxide moiety attached to theN-terminus of the IL-28 or IL-29 moiety. PEG is one suitable polyalkyloxide. As an illustration, IL-28 or IL-29 can be modified with PEG, aprocess known as “PEGylation.” PEGylation of IL-28 or IL-29 can becarried out by any of the PEGylation reactions known in the art (see,for example, EP 0 154 316, Delgado et at., Critical Reviews inTherapeutic Drug Carrier Systems 9:249 (1992), Duncan and Spreafico,Clin. Pharmacokinet. 27:290 (1994), and Francis et al, Int J Hematol68:1 (1998)). The methods of the present invention includeadministration of IL-28, IL-29, and mutants thereof conjugated towater-soluble polymers, such as PEG.

IL-28A, IL-29, IL-28B, zcyto24 and zcyto25, each have been shown to forma complex with the orphan receptor designated zcytor19 (IL-28RA).IL-28RA is described in a commonly assigned patent applicationPCT/US01/44808. IL-28B, IL-29, zcyto24, and zcyto25 have been shown tobind or signal through IL-28RA as well, further supporting that IL-28A,IL-29, IL-28B, zcyto24 and zcyto25 are members of the same family ofcytokines. IL-28RA receptor is a class II cytokine receptor. Class IIcytokine receptors usually bind to four-helix-bundle cytokines. Forexample, interleukin-10 and the interferons bind receptors in this class(e.g., interferon-gamma receptor, alpha and beta chains and theinterferon-alpha/beta receptor alpha and beta chains).

Class II cytokine receptors are characterized by the presence of one ormore cytokine receptor modules (CRM) in their extracellular domains.Other class II cytokine receptors include zcytor11 (commonly owned U.S.Pat. No. 5,965,704), CRF2-4 (Genbank Accession No. Z17227), IL-10R(Genbank Accession No.s U00672 and NM_(—)001558), DIRS1, zcytor7(commonly owned U.S. Pat. No. 5,945,511), and tissue factor. IL-28RA,like all known class II receptors except interferon-alpha/beta receptoralpha chain, has only a single class II CRM in its extracellular domain.

Analysis of a human cDNA clone encoding IL-28RA (SEQ ID NO:11) revealedan open reading frame encoding 520 amino acids (SEQ ID NO:12) comprisinga secretory signal sequence (residues 1 (Met) to 20 (Gly) of SEQ IDNO:12) and a mature IL-28RA cytokine receptor polypeptide (residues 21(Arg) to 520 (Arg) of SEQ ID NO:12) an extracellular ligand-bindingdomain of approximately 206 amino acid residues (residues 21 (Arg) to226 (Asn) of SEQ ID NO:12), a transmembrane domain of approximately 23amino acid residues (residues 227 (Trp) to 249 (Trp) of SEQ ID NO:12),and an intracellular domain of approximately 271 amino acid residues(residues 250 (Lys) to 520 (Arg) of SEQ ID NO:12). Within theextracellular ligand-binding domain, there are two fibronectin type IIIdomains and a linker region. The first fibronectin type III domaincomprises residues 21 (Arg) to 119 (Tyr) of SEQ ID NO:12, the linkercomprises residues 120 (Leu) to 124 (Glu) of SEQ ID NO:12, and thesecond fibronectin type III domain comprises residues 125 (Pro) to 223(Pro) of SEQ ID NO:12.

In addition, a human cDNA clone encoding a IL-28RA variant with a 29amino acid deletion was identified. This IL-28RA variant (as shown inSEQ ID NO:13) comprises an open reading frame encoding 491 amino acids(SEQ ID NO:14) comprising a secretory signal sequence (residues 1 (Met)to 20 (Gly) of SEQ ID NO:14) and a mature IL-28RA cytokine receptorpolyptide (residues 21 (Arg) to 491 (Arg) of SEQ ID NO:14) anextracellular ligand-binding domain of approximately 206 amino acidresidues (residues 21 (Arg) to 226 (Asn) of SEQ ID NO:14, atransmembrane domain of approximately 23 amino acid residues (residues227 (Trp) to 249 (Trp) of SEQ ID NO:14), and an intracellular domain ofapproximately 242 amino acid residues (residues 250 (Lys) to 491 (Arg)of SEQ ID NO:14).

A truncated soluble form of the IL-28RA receptor mRNA appears to benaturally expressed. Analysis of a human cDNA alone encoding thetruncated soluble IL-28RA (SEQ ID NO:15) revealed an open reading frameencoding 211 amino acids (SEQ ID NO:16) comprising a secretory signalsequence (residues 1 (Met) to 20 (Gly) of SEQ ID NO:16) and a maturetruncated soluble IL-28RA receptor polyptide (residues 21 (Arg) to 211(Ser) of SEQ ID NO:16) a truncated extracellular ligand-binding domainof approximately 143 amino acid residues (residues 21 (Arg) to 163 (Trp)of SEQ ID NO:16), no transmembrane domain, but an additional domain ofapproximately 48 amino acid residues (residues 164 (Lys) to 211 (Ser) ofSEQ ID NO:16).

IL-28RA is a member of the same receptor subfamily as the class IIcytokine receptors, and receptors in this subfamily may associate toform homodimers that transduce a signal. Several members of thesubfamily (e.g., receptors that bind interferon, IL-10, IL-19, andIL-TIF) combine with a second subunit (termed a β-subunit) to bindligand and transduce a signal. However, in many cases, specificβ-subunits associate with a plurality of specific cytokine receptorsubunits. For example, class II cytokine receptors, such as, zcytor11(U.S. Pat. No. 5,965,704) and CRF2-4 receptor heterodimerize to bind thecytokine IL-TIF (See, WIPO publication WO 00/24758; Dumontier et al., J.Immunol. 164:1814-1819, 2000; Spencer, S D et al., J. Exp. Med.187:571-578, 1998; Gibbs, V C and Pennica Gene 186:97-101, 1997 (CRF2-4cDNA); Xie, M H et al., J. Biol. Chem. 275:31335-31339, 2000). IL-10βreceptor is believed to be synonymous with CRF2-4 (Dumoutier, L. et al.,Proc. Nat'l. Acad. Sci. 97:10144-10149, 2000; Liu Y et al, J Immunol.152; 1821-1829, 1994 (IL-10R cDNA). Therefore, one could expect thatzcyto20, zcyto21, zcyto22, zcyto24 and zcyto25 would bind eithermonomeric, homodimeric, heterodimeric and multimeric zcytor19 receptors.Experimental evidence has identified CRF2-4 as the putative bindingpartner for IL-28RA.

Examples of biological activity for molecules used to identify IL-28 orIL-29 molecules that are useful in the methods of the present inventioninclude molecules that can bind to the IL-28 receptor with somespecificity. Generally, a ligand binding to its cognate receptor isspecific when the K_(D) falls within the range of 100 nM to 100 pM.Specific binding in the range of 100 mM to 10 nM K_(D) is low affinitybinding. Specific binding in the range of 2.5 pM to 100 pM K_(D) is highaffinity binding. In another example, biologically active IL-28 or IL-29molecules are capable of some level of antiviral activity associatedwith wildtype I L-28 or IL-29.

Use of IL-28A, IL-28B, and IL-29 for Viral Infections.

IL-28 and IL-29 can be used in treating liver specific diseases, inparticular liver disease where viral infection is in part an etiologicagent. In particular IL-28 and IL-29 will be used to treat a mammal witha viral infection selected from the group consisting of hepatitis A,hepatitis B, hepatitis C, and hepatitis D. When liver disease isinflammatory and continuing for at least six months, it is generallyconsidered chronic hepatitis. Hepatitis C virus (HCV) patients activelyinfected will be positive for HCV-RNA in their blood, which isdetectable by reverse transcritptase/polymerase chain reaction (RE-PCR)assays. The methods of the present invention will slow the progressionof the liver disease. Clinically, diagnostic tests for HCV includeserologic assays for antibodies and molecular tests for viral particles.Enzyme immunoassays are available (Vrielink et al., Transfusion37:845-849, 1997), but may require confirmation using additional testssuch as an immunoblot assay (Pawlotsky et al., Hepatology 27:1700-1702,1998). Qualitative and quantitative assays generally use polymerasechain reaction techniques, and are preferred for assessing viremia andtreatment response (Poynard et al., Lancet 352:1426-1432, 1998;McHutchinson et al., N. Engl. J. Med. 339:1485-1492, 1998). Severalcommercial tests are available, such as, quantitative RE-PCR (AmplicorHCV Monitor™, Roche Molecular Systems, Branchburg, N.J.) and a branchedDNA (deoxyribonucleic acid) signal amplification assay (Quantiplex™ HCVRNA Assay [bDNA], Chiron Corp., Emeryville, Calif.). A non-specificlaboratory test for liver inflammation or necrosis measures alanineaminotransferase level (ALT) and is inexpensive and readily available(National Institutes of Health Consensus Development Conference Panel,Hepatology 26 (Suppl. 1):2S-10S, 1997). Histologic evaluation of liverbiopsy is generally considered the most accurate means for determininghepatitis progression (Yano et al., Hepatology 23:1334-1340, 1996.) Fora review of clinical tests for HCV, see, Lauer et al., N. Engl. J. Med.345:41-52, 2001.

There are several in vivo models for testing HBV and HCV that are knownto those skilled in art. For example, the effects of IL-28 or IL-29 onmammals infected with HBV can accessed using a woodchuck model. Briefly,woodchucks chronically infected with woodchuck hepatitis virus (WHV)develop hepatitis and hepatocellular carcinoma that is similar todisease in humans chronically infected with HBV. The model has been usedfor the preclinical assessment of antiviral activity. A chronicallyinfected WHV strain has been established and neonates are inoculatedwith serum to provide animals for studying the effects of certaincompounds using this model. (For a review, see, Tannant et al., ILAR J.42 (2):89-102, 2001). Chimpanzees may also be used to evaluate theeffect of IL-28 and IL-29 on HBV infected mammals. Using chimpanzees,characterization of HBV was made and these studies demonstrated that thechimpanzee disease was remarkably similar to the disease in humans(Barker et al., J. Infect. Dis. 132:451-458, 1975 and Tabor et al., J.Infect. Dis. 147:531-534, 1983.) The chimpanzee model has been used inevaluating vaccines (Prince et al., In Vaccines 97 Cold Spring HarborLaboratory Press, 1997.) Therapies for HIV are routinely tested usingnon-human primates infected with simian immunodeficiency viruses (for areview, see, Hirsch et al., Adv. Pharmcol. 49:437-477, 2000 andNathanson et al., AIDS 13 (suppl. A):S113-S120, 1999.) For a review ofuse of non-human primates in HIV, hepatitis, malaria, respiratorysyncytial virus, and other diseases, see, Sibal et al., ILAR J. 42(2):74-84, 2001.

Other examples of the types of viral infections for which IL-28A,IL-28B, and IL-29 can be useful include, but are not limited to:infections caused by DNA Viruses (e.g., Herpes Viruses such as HerpesSimplex viruses, Epstein-Barr virus, Cytomegalovirus; Pox viruses suchas Variola (small pox) virus; Hepadnaviruses (e.g, Hepatitis B virus);Papilloma viruses; Adenoviruses); RNA Viruses (e.g., HIV I, II; HTLV I,II; Poliovirus; Hepatitis A; Orthomyxoviruses (e.g., Influenza viruses);Paramyxoviruses (e.g., Measles virus); Rabies virus; Hepatitis C);Rhinovirus, Respiratory Syncytial Virus, West Nile Virus, Yellow Fever,Rift Vallley Virus, Lassa Fever Virus, Ebola Virus, LymphocyticChoriomeningitis Virus, which replicates in tissues including liver, andthe like. Moreover, examples of the types of diseases for which IL-28and IL-29 could be used include, but are not limited to: Acquiredimmunodeficiency; Hepatitis; Gastroenteritis; Hemorrhagic diseases;Enteritis; Carditis; Encephalitis; Paralysis; Brochiolitis; Upper andlower respiratory disease; Respiratory Papillomatosis; Arthritis;Disseminated disease, hepatocellular carcinoma resulting rom chronicHepatitis C infection. In addition, viral disease in other tissues maybe treated with IL-28A, IL-28B, and IL-29, for example viral meningitis,and HIV-related disease. For example, a transgenic model for testing theactivity of a therapeutic sample is described in the following examplesand described in Morrey, et al., Antiviral Ther., 3 (Suppl 3):59-68,1998.

Animal models that are used to test for efficacy in specific viruses areknown. For example, Dengue Virus can be tested using a model as such asdescribed in Huang et al., J. Gen. Virol. September;81(Pt9):2177-82,2000. West Nile Virus can be tested using the model as described in Xiaoet al., Emerg. Infect. Dis. July-August;7(4):714-21, 2001or Mashimo etal., Proc. Natl. Acad. Sci. USA. August 20;99(17):11311-6, 2002.Venezuelan equine encephalitis virus model is described in Jackson etal., Veterinary Pathology, 28 (5):410-418, 1991; Vogel et al., Arch.Pathol. Lab. Med. February;120(2):164-72, 1996; Lukaszewski and Brooks,J. of Virology, 74(11):5006-5015, 2000. Rhinoviruses models aredescribed in Yin and Lomax, J. Gen. Virol. 67 (Pt11):2335-40, 1986.Models for respiratory syncytial virus are described in Byrd and Prince,Clin. Infect. Dis. 25(6):1363-8, 1997. Other models are known in the artand it is well within the skill of those ordinarily skilled in the artto know how to use such models.

IL-28A, IL-28B, and IL-29 can be used in combination with antiviralagents, including those described above. Some of the more commontreatments for viral infection include drugs that inhibit viralreplication such as ACYCLOVIR™. In addition, the combined use of some ofthese agents form the basis for highly active antiretroviral therapy(HAART) used for the treatment of AIDS. Examples in which thecombination of immunotherapy (i.e. cytokines) and antiviral drugs showsimproved efficacy include the use of interferon plus RIBAVIRIN™ for thetreatment of chronic hepatitis C infection (Maddrey, Semin. Liver.Dis.19 Suppl 1:67-75, 1999) and the combined use of IL-2 and HAART(Ross, et al, ibid.) Thus, as IL-28 and IL-29 can stimulate the immunesystem against disease, it can similarly be used in HAART applications.

In particular, IL-28A, IL-28B, and IL-29 may be useful in monotherapy orcombination therapy with IFN-α (with or without RIBAVIRIN™) in patientswho do not respond well to IFN therapy. These patients may not respondto IFN therapy due to having less type 1 interferon receptor on thesurface of their cells (Yatsuhashi H, et al., J Hepatol. June30(6):995-1003, 1999; Mathai et al., J Interferon Cytokine Res.September 19(9):1011-8, 1999; Fukuda et al., J Med. Virol. 63(3):220-7,2001). IL-28A, IL-28B, and IL-29 may also be useful in monotherapy orcombination therapy with IFN-α (with or without RIBAVIRIN™) in patientswho have less type I interferon receptor on the surface of their cellsdue to down-regulation of the type I interferon receptor after type Iinterferon treatment (Dupont et al., J. Interferon Cytokine Res.22(4):491-501, 2002).

IL-28 or IL-29 can be used in combination with other immunotherapiesincluding cytokines, immunoglobulin transfer, and various co-stimulatorymolecules. In addition to antiviral drugs, IL-28, IL-29, or mutantsthereof could be used in combination with any other immunotherapy thatis intended to stimulate the immune system. Thus, IL-28, IL-29, ormutants thereof could be used with other cytokines such as Interferon orIL-2. IL-28, IL-29, or mutants thereof could also be added to methods ofpassive immunization that involve immunoglobulin transfer, one examplebring the use of antibodies to treat RSV infection in high risk patients(Meissner H C, ibid.). In addition, IL-28, IL-29, or mutants thereofcould be used with additional co-stimulatory molecules such as 4-1BBligand that recognize various cell surface molecules like CD137 (Tan, JT et al., J Immunol. 163:4859-68, 1999).

C. Use of IL-28A, IL-28B, and IL-29 in Immunocompromised Patients

IL-28 and IL-29 can be used as a monotherapy for acute and chronic viralinfections and for immunocompromised patients. Methods that enhanceimmunity can accelerate the recovery time in patients with unresolvedinfections. Immunotherapies can have an even greater impact on subsetsof immunocompromised patients such as the very young or elderly as wellas patients that suffer immunodeficiencies acquired through infection,or induced following medical interventions such as chemotherapy or bonemarrow ablation. Examples of the types of indications being treated viaimmune-modulation include; the use of IFN-α for chronic hepatitis (PerryC M, and Jarvis B, Drugs 61:2263-88, 2001), the use of IL-2 followingHIV infection (Mitsuyasu R., J. Infect. Dis. 185 Suppl 2:S115-22, 2002;and Ross R W et al., Expert Opin. Biol. Ther. 1:413-24, 2001), and theuse of either IFN-α (Faro A, Springer Semin. Immunopathol.20:425-36,1998) for treating Epstein Barr Virus infections followingtransplantation. Experiments performed in animal models indicate thatIL-2 and GM-CSF may also be efficacious for treating EBV relateddiseases (Baiocchi R A et al., J Clin. Invest. 108:887-94, 2001).

In summary, IL-28, IL-29, or mutants thereof can be used:

as a monotherapy for acute and chronic viral infections and forimmunocompromised patients. Methods that enhance immunity can acceleratethe recovery time in patients with unresolved infections.

in combination with other antiviral agents such as ACYCLOVIR™,interferon plus RIBAVIRIN™.

in combination with other immunotherapies including cytokines,immunoglobulin transfer, and various co-stimulatory molecules.

to treat a mammal with a chronic or acute viral infection that hasresulted liver inflammation, thereby reducing the viral infection and/orliver inflammation. In particular IL-28 and IL-29 will be used to treata mammal with a viral infection selected from the group consisting ofhepatitis A, hepatitis B, hepatitis C, and hepatitis D.

as an antiviral agent in viral infections selected from the groupconsisting of respiratory syncytial virus, herpes virus, Epstein-Barrvirus, influenza virus, adenovirus, parainfluenza virus, rhino virus,coxsackie virus, vaccinia virus, west nile virus, dengue virus,Venezuelan equine encephalitis virus, pichinde virus and polio virus.

EXAMPLES Example 1

Induction of IL-28A, IL-29 IL-28B by Poly I:C and Viral Infection

Freshly isolated human peripheral blood mononuclear cells were grown inthe presence of polyinosinic acid-polycytidylic acid (poly I:C; 100μg/ml) (SIGMA; St. Louis, Mo.), encephalomyocarditis virus (EMCV) withan MOI of 0. 1, or in medium alone. After a 15 h incubation, total RNAwas isolated from cells and treated with RNase-free DNase. 100 ng totalRNA was used as template for one-step RE-PCR using the SuperscriptOne-Step RE-PCR with Platinum Taq kit and gene-specific primers assuggested by the manufacturer (Invitrogen).

Low to undetectable amounts of human IL-28A, IL-28B, and IL-29, IFN-αand IFN-β RNA were seen in untreated cells. In contrast, the amount ofIL-28A, IL-29, IL-28B RNA was increased by both poly I:C treatment andviral infection, as was also seen for the type I interferons. Theseexperiments indicate that IL-28A, IL-29, IL-28B, like type Iinterferons, can be induced by double-stranded RNA or viral infection.

Example 2

IL-28 IL-29 Signaling Activity Compared to IFNα in HepG2 Cells.

A. Cell Transfections

HepG2 cells were transfected as follows:700,000 HepG2 cells/well (6 wellplates) were plated approximately 18 h prior to transfection in 2milliliters DMEM+10% fetal bovine serum. Per well, 1 microgrampISRE-Luciferase DNA (Stratagene) and 1 microgram pIRES2-EGFP DNA(Clontech,) were added to 6 microliters Fugene 6 reagent (RocheBiochemicals) in a total of 100 microliters DMEM. This transfection mixwas added 30 minutes later to the pre-plated HepG2 cells. Twenty-fourhours later the transfected cells were removed from the plate usingtrypsin-EDTA and replated at approximately 25,000 cells/well in 96 wellmicrotiter plates. Approximately 18 h prior to ligand stimulation, mediawas changed to DMEM+0.5% FBS.

B. Signal Transduction Reporter Assays

The signal transduction reporter assays were done as follows: Followingan 18 h incubation at 37° C. in DMEM+0.5% FBS, transfected cells werestimulated with 100 ng/ml IL-28A, IL-29, IL-28B, zcyto24, zcyto25 andhuIFN-α2a ligands. Following a 4-hour incubation at 37° degrees, thecells were lysed, and the relative light units (RLU) were measured on aluminometer after addition of a luciferase substrate. The resultsobtained are shown as the fold induction of the RLU of the experimentalsamples over the medium alone control (RLU of experimental samples/RLUof medium alone=fold induction). Table 2 shows that IL-28A, IL-29,IL-28B, zcyto24 and zcyto25 induce ISRE signaling in human HepG2 livercells transfected with ISRE-luciferase. TABLE 2 Fold Induction ofCytokine-dependent ISRE Signaling in HepG2 Cells Cytokine Fold Inductn.IL-28A 5.6 IL-29 4 IL-28B 5.8 Zcyto24 4.7 Zcyto25 3 HuIFN-a2a 5.8

Example 3

IL-29 Antiviral Activity Compared to IFNα in HepG2 Cells.

An antiviral assay was adapted for EMCV (American Type CultureCollection # VR-129B, Manassas, Va.) with human cells (Familletti, P.,et al., Methods Enzym. 78:387-394, 1981). Cells were plated withcytokines and incubated 24 hours prior to challenge by EMCV at amultiplicity of infection of 0.1 to 1. The cells were analyzed forviability with a dye-uptake bioassay 24 hours after infection (Berg, K.,et al., Apmis 98:156-162, 1990 ). Target cells were given MTT andincubated at 37° C. for 2 hours. A solubiliser solution was added,incubated overnight at 37° C. and the optical density at 570 nm wasdetermined. OD570 is directly proportional to antiviral activity.

The results show the antiviral activity when IL-29 and IFN on weretested with HepG2 cells: IL-29, IFN-β and IFN α-2a were added at varyingconcentration to HepG2 cells prior to EMCV infection and dye-uptakeassay. The mean and standard deviation of the OD570 from triplicatewells is plotted. OD570 is directly proportional to antiviral activity.For IL-29, the EC50 was 0.60 ng/ml; for IFN-α2a, the EC50 was 0.57ng/ml; and for IFN-β, the EC50 was 0.46 ng/ml.

Example 4

IL-28RA mRNA Expression in Liver and Lymphocyte Subsets.

In order to further examine the mRNA distribution for IL-28RA,semi-quantitative RE-PCR was performed using the SDS 7900HT system(Applied Biosystems, Calif.). One-step RE-PCR was performed using 100 ngtotal RNA for each sample and gene-specific primers. A standard curvewas generated for each primer set using Bjab RNA and all sample valueswere normalized to HPRT. The normalized results are summarized in Tables2-4. The normalized values for IFNAR2 and CRF2-4 are also shown.

Table 3: B and T cells express significant levels of IL-28RA mRNA. Lowlevels are seen in dendritic cells and most monocytes. TABLE 3Cell/Tissue IL-28RA IFNAR2 CRF2-4 Dendritic Cells unstim .04 5.9 9.8Dendritic Cells + IFNg .07 3.6 4.3 Dendritic Cells .16 7.85 3.9 CD14+stim'd with LPS/IFNg .13 12 27 CD14+ monocytes resting .12 11 15.4 HuCD14+ Unact. 4.2 TBD TBD Hu CD14+ 1 ug/ml LPS act. 2.3 TBD TBD H.Inflamed tonsil 3 12.4 9.5 H. B-cells + PMA/Iono 4 & 24 hrs 3.6 1.3 1.4Hu CD19+ resting 6.2 TBD TBD Hu CD19+ 4 hr. PMA/Iono 10.6 TBD TBD HuCD19+ 24 hr Act. PMA/Iono 3.7 TBD TBD IgD+ B-cells 6.47 13.15 6.42 IgM+B-cells 9.06 15.4 2.18 IgD− B-cells 5.66 2.86 6.76 NKCells + PMA/Iono 06.7 2.9 Hu CD3+ Unactivated 2.1 TBD TBD CD4+ resting .9 8.5 29.1 CD4+Unstim 18 hrs 1.6 8.4 13.2 CD4+ + Poly I/C 2.2 4.5 5.1 CD4+ + PMA/Iono.3 1.8 .9 CD3 neg resting 1.6 7.3 46 CD3 neg unstim 18 hrs 2.4 13.2 16.8CD3 neg + Poly I/C 18 hrs 5.7 7 30.2 CD3 neg + LPS 18 hrs 3.1 11.9 28.2CD8+ unstim 18 hrs 1.8 4.9 13.1 CD8+ stim'd with PMA/Ion 18 hrs .3 .61.1

As shown in Table 4, normal liver tissue and liver derived cell linesdisplay substantial levels of IL-28RA and CRF2-4 mRNA. TABLE 4Cell/Tissue IL-28RA IFNAR2 CRF2-4 HepG2 1.6 3.56 2.1 HepG2 UGAR May 10,2002 1.1 1.2 2.7 HepG2, CGAT HKES081501C 4.3 2.1 6 HuH7 May 10, 20021.63 16 2 HuH7 hepatoma - CGAT 4.2 7.2 3.1 Liver, normal - 11.7 3.2 8.4CGAT #HXYZ020801K Liver, NAT - Normal adjacent tissue 4.5 4.9 7.7 Liver,NAT - Normal adjacent tissue 2.2 6.3 10.4 Hep SMVC hep vein 0 1.4 6.5Hep SMCA hep. Artery 0 2.1 7.5 Hep. Fibro 0 2.9 6.2 Hep. Ca. 3.8 2.9 5.8Adenoca liver 8.3 4.2 10.5 SK-Hep-1 adenoca. Liver .1 1.3 2.5 AsPC-1 Hu.Pancreatic adenocarc. .7 .8 1.3 Hu. Hep. Stellate cells .025 4.4 9.7

As shown in Table 5, primary airway epithelial cells contain abundantlevels of IL-28RA and CRF2-4. TABLE 5 Cell/Tissue IL-28RA IFNAR2 CRF2-4U87MG - glioma 0  .66  .99 NHBE unstim 1.9 1.7 8.8 NHBE + TNF-alpha 2.25.7 4.6 NHBE + poly I/C 1.8 nd nd Small Airway Epithelial Cells 3.9 3.327.8  NHLF - Normal human lung fibroblasts 0 nd nd

As shown in Table 6, IL-28RA is present in normal and diseased liverspecimens, with increased expression in tissue from Hepatitis C andHepatitis B infected specimens. TABLE 6 Cell/Tissue IL-28RA CRF2-4IFNAR2 Liver with Coagulation Necrosis 8.87 15.12 1.72 Liver withAutoimmune Hepatitis 6.46 8.90 3.07 Neonatal Hepatitis 6.29 12.46 6.16Endstage Liver disease 4.79 17.05 10.58 Fulminant Liver Failure 1.9014.20 7.69 Fulminant Liver failure 2.52 11.25 8.84 Cirrhosis, primarybiliary 4.64 12.03 3.62 Cirrhosis Alcoholic (Laennec's) 4.17 8.30 4.14Cirrhosis, Cryptogenic 4.84 7.13 5.06 Hepatitis C+, with cirrhosis 3.647.99 6.62 Hepatitis C+ 6.32 11.29 7.43 Fulminant hepatitis secondary toHep A 8.94 21.63 8.48 Hepatitis C+ 7.69 15.88 8.05 Hepatitis B+ 1.6112.79 6.93 Normal Liver 8.76 5.42 3.78 Normal Liver 1.46 4.13 4.83 LiverNAT 3.61 5.43 6.42 Liver NAT 1.97 10.37 6.31 Hu Fetal Liver 1.07 4.873.98 Hepatocellular Carcinoma 3.58 3.80 3.22 Adenocarcinoma Liver 8.3010.48 4.17 hep. SMVC, hep. Vein 0.00 6.46 1.45 Hep SMCA hep. Artery 0.007.55 2.10 Hep. Fibroblast 0.00 6.20 2.94 HuH7 hepatoma 4.20 3.05 7.24HepG2 Hepatocellular carcinoma 3.40 5.98 2.11 SK-Hep-1 adenocar. Liver0.03 2.53 1.30 HepG2 Unstim 2.06 2.98 2.28 HepG2 + zcyto21 2.28 3.012.53 HepG2 + IFNα 2.61 3.05 3.00 Normal Female Liver - degraded 1.386.45 4.57 Normal Liver - degraded 1.93 4.99 6.25 Normal Liver - degraded2.41 2.32 2.75 Disease Liver - degraded 2.33 3.00 6.04 PrimaryHepatocytes from Clonetics 9.13 7.97 13.30

As shown in Tables 7-11, IL-28RA is detectable in normal B cells, Blymphoma cell lines, T cells, T lymphoma cell lines (Jurkat), normal andtransformed lymphocytes (B cells and T cells) and normal humanmonocytes. TABLE 7 HPRT IL-28RA IL-28RA IFNR2 CRF2-4 Mean Mean normIFNAR2 norm CRF2-4 Norm CD14+ 24 hr unstim #A38 13.1 68.9 5.2 92.3 7.0199.8 15.2 CD14+ 24 hr stim #A38 6.9 7.6 1.1 219.5 31.8 276.6 40.1 CD14+24 hr unstim #A112 17.5 40.6 2.3 163.8 9.4 239.7 13.7 CD14+ 24 hr stim#A112 11.8 6.4 0.5 264.6 22.4 266.9 22.6 CD14+ rest #X 32.0 164.2 5.11279.7 39.9 699.9 21.8 CD14+ + LPS #X 21.4 40.8 1.9 338.2 15.8 518.024.2 CD14+ 24 hr unstim #A39 26.3 86.8 3.3 297.4 11.3 480.6 18.3 CD14+24 hr stim #A39 16.6 12.5 0.8 210.0 12.7 406.4 24.5 HL60 Resting 161.20.2 0.0 214.2 1.3 264.0 1.6 HL60 + PMA 23.6 2.8 0.1 372.5 15.8 397.516.8 U937 Resting 246.7 0.0 0.0 449.4 1.8 362.5 1.5 U937 + PMA 222.7 0.00.0 379.2 1.7 475.9 2.1 Jurkat Resting 241.7 103.0 0.4 327.7 1.4 36.10.1 Jurkat Activated 130.7 143.2 1.1 Colo205 88.8 43.5 0.5 HT-29 26.530.5 1.2

TABLE 8 HPRT SD IL-28RA SD Mono 24 hr unstim #A38 0.6 2.4 Mono 24 hrstim #A38 0.7 0.2 Mono 24 hr unstim 2.0 0.7 #A112 Mono 24 hr stim #A1120.3 0.1 Mono rest #X 5.7 2.2 Mono + LPS #X 0.5 1.0 Mono 24 hr unstim#A39 0.7 0.8 Mono 24 hr stim #A39 0.1 0.7 HL60 Resting 19.7 0.1 HL60 +PMA 0.7 0.4 U937 Resting 7.4 0.0 U937 + PMA 7.1 0.0 Jurkat Resting 3.71.1 Jurkat Activated 2.4 1.8 Colo205 1.9 0.7 HT-29 2.3 1.7

TABLE 9 Mean Mean Mean IL- Mean Hprt IFNAR2 28RA CRF CD3+/CD4+ 0 10.185.9 9.0 294.6 CD4/CD3+ Unstim 18 hrs 12.9 108.7 20.3 170.4 CD4+/CD3++Poly I/C 18 hrs 24.1 108.5 52.1 121.8 CD4+/CD3+ + PMA/Iono 18 hrs 47.883.7 16.5 40.8 CD3 neg 0 15.4 111.7 24.8 706.1 CD3 neg unstim 18 hrs15.7 206.6 37.5 263.0 CD3 neg + Poly I/C 18 hrs 9.6 67.0 54.7 289.5 CD3neg + LPS 18 hrs 14.5 173.2 44.6 409.3 CD8+ Unstim. 18 hrs 6.1 29.7 11.179.9 CD8+ + PMA/Iono 18 hrs 78.4 47.6 26.1 85.5 12.8.1 - NHBE Unstim47.4 81.1 76.5 415.6 12.8.2 - NHBE + TNF-alpha 42.3 238.8 127.7 193.9SAEC 15.3 49.9 63.6 426.0

TABLE 10 IL-28RA CRF IFNAR2 IL-28RA CRF IFNAR2 Norm Norm Norm SD SD SDCD3+/CD4+ 0 0.9 29.1 8.5 0.1 1.6 0.4 CD4/CD3+ Unstim 18 hrs 1.6 13.2 8.40.2 1.6 1.4 CD4+/CD3+ + Poly I/C 18 hrs 2.2 5.1 4.5 0.1 0.3 0.5CD4+/CD3+ + PMA/Iono 18 hrs 0.3 0.9 1.8 0.0 0.1 0.3 CD3 neg 0 1.6 46.07.3 0.2 4.7 1.3 CD3 neg unstim 18 hrs 2.4 16.8 13.2 0.4 2.7 2.3 CD3neg + Poly I/C 18 hrs 5.7 30.2 7.0 0.3 1.7 0.8 CD3 neg + LPS 18 hrs 3.128.2 11.9 0.4 5.4 2.9 CD8+ Unstim. 18 hrs 1.8 13.1 4.9 0.1 1.1 0.3CD8+ + PMA/Iono 18 hrs 0.3 1.1 0.6 0.0 0.1 0.0 12.8.1 - NHBE Unstim 1.68.8 1.7 0.1 0.4 0.1 12.8.2 - NHBE + TNF-alpha 3.0 4.6 5.7 0.1 0.1 0.1SAEC 4.1 27.8 3.3 0.2 1.1 0.3

TABLE 11 SD SD IL- SD SD Hprt IFNAR2 28RA CRF CD3+/CD4+ 0 0.3 3.5 0.612.8 CD4/CD3+ Unstim 18 hrs 1.4 13.7 1.1 8.5 CD4+/CD3+ +Poly I/C 18 hrs1.3 9.8 1.6 3.4 CD4+/CD3+ + PMA/Iono 18 hrs 4.0 10.3 0.7 3.7 CD3 neg 01.4 16.6 1.6 28.6 CD3 neg unstim 18 hrs 2.4 16.2 2.7 12.6 CD3 neg + PolyI/C 18 hrs 0.5 7.0 1.0 8.3 CD3 neg + LPS 18 hrs 1.0 39.8 5.6 73.6 CD8+Unstim. 18 hrs 0.2 1.6 0.5 6.1 CD8+ + PMA/Iono 18 hrs 1.3 1.7 0.2 8.112.8.1 - NHBE Unstim 2.4 5.6 2.7 2.8 12.8.2 - NHBE + TNF-alpha 0.5 3.43.5 3.4 SAEC 0.5 4.8 1.8 9.9

Example 5

Mouse IL-28 Does Not Effect Daudi Cell Proliferation

Human Daudi cells were suspended in RPMI+10% FBS at 50,000cells/milliliter and 5000 cells were plated per well in a 96 well plate.IL-29-CEE (IL-29 conjugated with glu tag), IFN-γ or IFN-α2a was added in2-fold serial dilutions to each well. IL-29-CEE was used at aconcentration range of from 1000 ng/ml to 0.5 ng/ml. IFN-γ was used at aconcentration range from 125 ng/ml to 0.06 ng/ml. IFN-α2a was used at aconcentration range of from 62 ng/ml to 0.03 ng/ml. Cells were incubatedfor 72 h at 37° C. After 72 h. Alamar Blue (Accumed, Chicago, Ill.) wasadded at 20 microliters/well. Plates were further incubated at 37° C.,5% CO, for 24 hours. Plates were read on the Fmax™ plate reader(Molecular Devices, Sunnyvale, Calif.) using the SoftMax™ Pro program,at wavelengths 544 (Excitation) and 590 (Emission). Alamar Blue gives afluourometric readout based on the metabolic activity of cells, and isthus a direct measurement of cell proliferation in comparison to anegative control. The results indicate that IL-29-CEE, in contrast toIFN-α2a, has no significant effect on proliferation of Daudi cells.

Example 6

Mouse IL-28 Does Not Have Antiproliferative Effect on Mouse B Cells

Mouse B cells were isolated from 2 Balb/C spleens (7 months old) bydepleting CD43+ cells using MACS magnetic beads. Purified B cells werecultured in vitro with LPS, anti-IgM or anti-CD40 monoclonal antibodies.Mouse IL-28 or mouse IFNα was added to the cultures and ³H-thymidine wasadded at 48 hrs. and ³H-thymidine incorporation was measured after 72hrs. culture.

IFNα at 10 ng/ml inhibited ³H-thymidine incorporation by mouse B cellsstimulated with either LPS or anti-IgM. However mouse IL-28 did notinhibit ³H-thymidine incorporation at any concentration tested including1000 ng/ml. In contrast, both mIFNα and mouse IL-28 increased ³Hthymidine incorporation by mouse B cells stimulated with anti-CD40 MAb.

These data demonstrate that mouse IL-28 unlike IFNα displays noantiproliferative activity even at high concentrations. In addition,zcyto24 enhances proliferation in the presence of anti-CD40 MAbs. Theresults illustrate that mouse IL-28 differs from IFNα in that mouseIL-28 does not display antiproliferative activity on mouse B cells, evenat high concentrations. In addition, mouse IL-28 enhances proliferationin the presence of anti-CD40 monoclonal antibodies.

Example 7

Bone Marrow Expansion Assay

Fresh human marrow mononuclear cells (Poietic Technologies,Gaithersburg, Md.) were adhered to plastic for 2 hrs in αMEM, 10% FBS,50 micromolar β-mercaptoethanol, 2 ng/ml FLT3L at 37° C. Non adherentcells were then plated at 25,000 to 45,000 cells/well (96 well tissueculture plates) in αMEM, 10% FBS, 50 micromolar β-mercaptoethanol, 2ng/ml FLT3L in the presence or absence of 1000 ng/ml IL-29-CEE, 100ng/ml IL-29-CEE, 10 ng/ml IL-29-CEE, 100 ng/ml IFN-α2a, 10 ng/ml IFN-α2aor 1 ng/ml IFN-α2a. These cells were incubated with a variety ofcytokines to test for expansion or differentiation of hematopoieticcells from the marrow (20 ng/ml IL-2, 2 ng/ml IL-3, 20 ng/ml IL-4, 20ng/ml IL-5, 20 ng/ml IL-7, 20 ng/ml IL-10, 20 ng/ml IL-i12, 20 ng/mlIL-15, 10 ng/ml IL-21 or no added cytokine). After 8 to 12 days AlamarBlue (Accumed, Chicago, Ill.) was added at 20 microliters/well. Plateswere further incubated at 37° C., 5% CO, for 24 hours. Plates were readon the Fmax™ plate reader (Molecular Devices Sunnyvale, Calif.) usingthe SoftMax™ Pro program, at wavelengths 544 (Excitation) and 590(Emission). Alamar Blue gives a fluourometric readout based on themetabolic activity of cells, and is thus a direct measurement of cellproliferation in comparison to a negative control.

IFN-α2a caused a significant inhibition of bone marrow expansion underall conditions tested. In contrast, IL-29 had no significant effect onexpansion of bone marrow cells in the presence of IL-3, IL-4, IL-5,IL-7, IL-10, IL-12, IL-21 or no added cytokine. A small inhibition ofbone marrow cell expansion was seen in the presence of IL-2 or IL-15.

Example 8

Inhibition of IL-28 and IL-29 Signaling With Soluble Receptor(zcytoR19/CRF2-4).

A. Signal Transduction Reporter Assay

A signal transduction reporter assay can be used to show the inhibitorproperties of zcytor19-Fc4 homodimeric and zcytor19-Fc/CRF2-4-Fcheterodimeric soluble receptors on zcyto20, zcyto21 and zcyto24signaling. Human embryonal kidney (HEK) cells overexpressing thezcytor19 receptor are transfected with a reporter plasmid containing aninterferon-stimulated response element (ISRE) driving transcription of aluciferase reporter gene. Luciferase activity following stimulation oftransfected cells with ligands (including zcyto20 (SEQ ID NO:18),zcyto2l (SEQ ID NO:20), zcyto24 (SEQ ID NO:8)) reflects the interactionof the ligand with soluble receptor.

B. Cell Transfections

293 HEK cells overexpressing zcytor19 were transfected as follows:700,000 293 cells/well (6 well plates) were plated approximately 18 hprior to transfection in 2 milliliters DMEM+ 10% fetal bovine serum. Perwell, 1 microgram pISRE-Luciferase DNA (Stratagene) and 1 microgrampIRES2-EGFP DNA (Clontech,) were added to 6 microliters Fugene 6 reagent(Roche Biochemicals) in a total of 100 microliters DMEM. Thistransfection mix was added 30 minutes later to the pre-plated 293 cells.Twenty-four hours later the transfected cells were removed from theplate using trypsin-EDTA and replated at approximately 25,000 cells/wellin 96 well microtiter plates. Approximately 18 h prior to ligandstimulation, media was changed to DMEM+0.5% FBS.

C. Signal Transduction Reporter Assays

The signal transduction reporter assays were done as follows: Followingan 18 h incubation at 37° C. in DMEM+0.5% FBS, transfected cells werestimulated with 10 ng/ml zcyto20, zcyto12or zcyto24 and 10 micrograms/mlof the following soluble receptors; human zcytor19-Fc homodimer, humanzcytor19-Fc/human CRF2-4-Fc heterodimer, human CRF2-4-Fc homodimer,murine zcytor19-Ig homodimer. Following a 4-hour incubation at 37° C.,the cells were lysed, and the relative light units (RLU) were measuredon a luminometer after addition of a luciferase substrate. The resultsobtained are shown as the percent inhibition of ligand-induced signalingin the presence of soluble receptor relative to the signaling in thepresence of PBS alone. Table 11 shows that the human zcytor19-Fc/humanCRF2-4 heterodimeric soluble receptor is able to inhibit zcyto20,zcyto21 and zcyto24-induced signaling between 16 and 45% of control. Thehuman zcytor19-Fc homodimeric soluble receptor is also able to inhibitzcyto21-induced signaling by 45%. No significant effects were seen withhuCRF2-4-Fc or muzcytor19-Ig homodimeric soluble receptors. TABLE 12Percent Inhibition of Ligand-induced Interferon Stimulated ResponseElement (ISRE) Signaling by Soluble Receptors Huzcytor19- Huzcytor19-Muzcytor19- Ligand Fc/huCRF2-4-Fc Fc HuCRF2-4-Fc Ig Zcyto20 16% 92% 80%91% Zcyto21 16% 45% 79% 103% Zcyto24 47% 90% 82% 89%

Example 9

IL-28 and IL-29 Inhibit HIV Replication in Fresh Human PBMCs

Human immunodeficiency virus (HIV) is a pathogenic retrovirus thatinfects cells of the immune system. CD4 T cells and monocytes are theprimary infected cell types. To test the ability of IL-28 and IL-29 toinhibit HIV replication in vitro, PBMCs from normal donors were infectedwith the HIV virus in the presence of IL-28, IL-29 andMetIL-29C172S-PEG.

Fresh human peripheral blood mononuclear cells (PBMCs) were isolatedfrom whole blood obtained from screened donors who were seronegative forHIV and HBV. Peripheral blood cells were pelleted/washed 2-3 times bylow speed centrifugation and resuspended in PBS to remove contaminatingplatelets. The washed blood cells were diluted 1:1 with Dulbecco'sphosphate buffered saline (D-PBS) and layered over 14 mL of LymphocyteSeparation Medium ((LSM; cellgro™ by Mediatech, Inc. Herndon, Va.);density 1.078±0.002 g/ml) in a 50 mL centrifuge tube and centrifuged for30 minutes at 600×G. Banded PBMCs were gently aspirated from theresulting interface and subsequently washed 2× in PBS by low speedcentrifugation. After the final wash, cells were counted by trypan blueexclusion and resuspended at 1×10⁷ cells/mL in RPMI 1640 supplementedwith 15% Fetal Bovine Serum (FBS), 2 mM L-glutamine, 4 μg/mL PHA-P. Thecells were allowed to incubate for 48-72 hours at 37° C. Afterincubation, PBMCs were centrifuged and resuspended in RPMI 1640 with 15%FBS, 2 mM L-glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin, 10μg/mL gentamycin, and 20 U/mL recombinant human IL-2. PBMCs weremaintained in the medium at a concentration of 1-2×10⁶ cells/mL withbiweekly medium changes until used in the assay protocol. Monocytes weredepleted from the culture as the result of adherence to the tissueculture flask.

For the standard PBMC assay, PHA-P stimulated cells from at least twonormal donors were pooled, diluted in fresh medium to a finalconcentration of 1×10⁶ cells/mL, and plated in the interior wells of a96 well round bottom microplate at 50 μL/well (5×10⁴ cells/well). Testdilutions were prepared at a 2× concentration in microtiter tubes and100 μL of each concentration was placed in appropriate wells in astandard format. IL-28, IL-29 and MetIL-29C172S-PEG were added atconcentrations from 0-10 μg/ml, usually in ½ log dilutions. 50 μL of apredetermined dilution of virus stock was placed in each test well(final MOI of 0.1). Wells with only cells and virus added were used forvirus control. Separate plates were prepared identically without virusfor drug cytotoxicity studies using an MTS assay system. The PBMCcultures were maintained for seven days following infection, at whichtime cell-free supernatant samples were collected and assayed forreverse transcriptase activity and p24 antigen levels.

A decrease in reverse transcriptase activity or p24 antigen levels withIL-28, IL-29 and MetIL-29C172S-PEG would be indicators of antiviralactivity. Result would demonstrate that IL-28 and IL-29 may havetherapeutic value in treating HIV and AIDS.

Example 10

IL-28 and IL-29 Inhibit GBV-B Replication in Marmoset Liver Cells

HCV is a member of the Flaviviridae family of RNA viruses. HCV does notreplicate well in either ex-vivo or in vitro cultures and therefore,there are no satisfactory systems to test the anti-HCV activity ofmolecules in vitro. GB virus B (GBV-B) is an attractive surrogate modelfor use in the development of anti-HCV antiviral agents since it has arelatively high level of sequence identity with HCV and is ahepatotropic virus. To date, the virus can only be grown in the primaryhepatocytes of certain non-human primates. This is accomplished byeither isolating hepatocytes in vitro and infecting them with GBV-B, orby isolating hepatocytes from GBV-B infected marmosets and directlyusing them with antiviral compounds.

The effects of IL-28, IL-29 and MetIL-29C172S-PEG are assayed on GBV-Bextracellular RNA production by TaqMan RE-PCR and on cytotoxicity usingCellTiter96® reagent (Promega, Madison, Wis.) at six half-log dilutionsIL-28, IL-29 or MetIL-29C172S-PEG polypeptide in triplicate. Untreatedcultures serve as the cell and virus controls. Both RIBAVIRIN® (200μg/ml at the highest test concentration) and IFN-α (5000 IU/ml at thehighest test) are included as positive control compounds. Primaryhepatocyte cultures are isolated and plated out on collagen-coatedplates. The next day the cultures are treated with the test samples(IL-28, IL-29, MetIL-29C172S-PEG, IFNα, or RIBAVIRIN®) for 24hr beforebeing exposed to GBV-B virions or treated directly with test sampleswhen using in vivo infected hepatocytes. Test samples and media areadded the next day, and replaced three days later. Three to four dayslater (at day 6-7 post test sample addition) the supernatant iscollected and the cell numbers quantitated with CellTiter96®. Viral RNAis extracted from the supernatant and quantified with triplicatereplicates in a quantitative TaqMan RE-PCR assay using an in vitrotranscribed RNA containing the RE-PCR target as a standard. The averageof replicate samples is computed. Inhibition of virus production isassessed by plotting the average RNA and cell number values of thetriplicate samples relative to the untreated virus and cell controls.The inhibitory concentration of drug resulting in 50% inhibition ofGBV-B RNA production (IC50) and the toxic concentration resulting indestruction of 50% of cell numbers relative to control values (TC50) arecalculated by interpolation from graphs created with the data.

Inhibition of the GBV-B RNA production by IL-28 and 29 is an indicationof the antiviral properties of IL-28 and IL-29 on this Hepatitis C-likevirus on hepatocytes, the primary organ of infection of Hepatitis C, andpositive results suggest that IL-28 or IL-29 may be useful in treatingHCV infections in humans.

Example 11

IL-28 IL-29 and MetIL-29C172S-PEG Inhibit HBV Replication in WT10 Cells

Chronic hepatitis B (HBV) is one of the most common and severe viralinfections of humans belonging to the Hepadnaviridae family of viruses.To test the antiviral activities of IL-28 and IL-29 against HBV, IL-28,IL-29 and MetIL-29C172S-PEG were tested against HBV in an in vitroinfection system using a variant of the human liver line HepG2. IL-28,IL-29 and MetIL-29C172S-PEG inhibited viral replication in this system,suggesting therapeutic value in treating HBV in humans.

WT10 cells are a derivative of the human liver cell line HepG2 2.2.15.WT10 cells are stably transfected with the HBV genome, enabling stableexpression of HBV transcripts in the cell line (Fu and Cheng,Antimicrobial Agents Chemother. 44(12):3402-3407, 2000). In theWT10assay the drug in question and a 3TC control will be assayed at fiveconcentrations each, diluted in a half-log series. The endpoints areTaqMan PCR for extracellular HBV DNA (IC50) and cell numbers usingCellTiter96 reagent (TC50). The assay is similar to that described byKorba et al. Antiviral Res. 15(3):217-228, 1991 and Korba et al.,Antiviral Res. 19(1):55-70, 1992. Briefly, WT10 cells are plated in96-well microtiter plates. After 16-24 hours the confluent monolayer ofHepG2-2.2.15 cells is washed and the medium is replaced with completemedium containing varying concentrations of a test samples intriplicate. 3TC is used as the positive control, while media alone isadded to cells as a negative control (virus control, VC). Three dayslater the culture medium is replaced with fresh medium containing theappropriately diluted test samples. Six days following the initialaddition of the test compound, the cell culture supernatant iscollected, treated with pronase and DNAse, and used in a real-timequantitative TaqMan PCR assay. The PCR-amplified HBV DNA is detected inreal-time by monitoring increases in fluorescence signals that resultfrom the exonucleolytic degradation of a quenched fluorescent probemolecule that hybridizes to the amplified HBV DNA. For each PCRamplification, a standard curve is simultaneously generated usingdilutions of purified HBV DNA. Antiviral activity is calculated from thereduction in HBV DNA levels (IC₅₀). A dye uptake assay is then employedto measure cell viability which is used to calculate toxicity (TC₅₀).The therapeutic index (TI) is calculated as TC₅₀/IC₅₀.

IL-28, IL-29 and MetIL-29C172S-PEG inhibited HepB viral replication inWT10 cells with an IC50<0.032μg/ml. This demonstrates antiviral activityof IL-28 and IL-29 against HBV grown in liver cell lines, providingevidence of therapeutic value for treating HBV in human patients.

Example 12

IL-28 IL-29 and MetIL-29C172S-PEG Inhibit BVDV Replication in BovineKidney Cells

HCV is a member of the Faviviridae family of RNA viruses. Other virusesbelonging to this family are the bovine viral diarrhea virus (BVDV) andyellow fever virus (YFV). HCV does not replicate well in either ex vivoor in vitro cultures and therefore there are no systems to test anti-HCVactivity in vitro. The BVDV and YFV assays are used as surrogate virusesfor HCV to test the antiviral activities against the Flavivirida familyof viruses.

The antiviral effects of IL-28, IL-29 and MetIL-29C172S-PEG wereassessed in inhibition of cytopathic effect assays (CPE). The assaymeasured cell death using Madin-Darby bovine kidney cells (MDBK) afterinfection with cytopathic BVDV virus and the inhibition of cell death byaddition of IL-28, IL-29 and MetIL-29C172S-PEG. The MDBK cells werepropagated in Dulbecco's modified essential medium (DMEM) containingphenol red with 10% horse serum, 1% glutamine and 1%penicillin-streptomycin. CPE inhibition assays were performed in DMEMwithout phenol red with 2% FBS, 1% glutamine and 1% Pen-Strep. On theday preceding the assays, cells were trypsinized (1% trypsin-EDTA),washed, counted and plated out at 10⁴ cells/well in a 96-wellflat-bottom BioCoat® plates (Fisher Scientific, Pittsburgh, Pa.) in avolume of 100 μl/well. The next day, the medium was removed and apre-titered aliquot of virus was added to the cells. The amount of viruswas the maximum dilution that would yield complete cell killing (>80%)at the time of maximal CPE development (day 7 for BVDV). Cell viabilitywas determined using a CellTiter96® reagent (Promega) according to themanufacturer's protocol, using a Vmax plate reader (Molecular Devices,Sunnyvale, Calif.). Test samples were tested at six concentrations each,diluted in assay medium in a half-log series. IFNα and RIBAVIRIN® wereused as positive controls. Test sample were added at the time of viralinfection. The average background and sample color-corrected data forpercent CPE reduction and percent cell viability at each concentrationwere determined relative to controls and the IC₅₀ calculated relative tothe TC₅₀.

IL-28, IL-29 and MetIL-29C172S-PEG inhibited cell death induced by BVDVin MDBK bovine kidney cells. IL-28 inhibited cell death with an IC₅₀ of0.02 μg/ml, IL-29 inhibited cell death with an IC₅₀ of 0.19 ∞g/ml, andMetIL-29C172S-PEG inhibited cell death with an IC₅₀ of 0.45 μg/ml. Thisdemonstrated that IL-28 and IL-29 have antiviral activity against theFlavivirida family of viruses.

Example 13

Induction of Interferon Stimulated Genes by IL-28 and IL-29

A. Human Peripheral Blood Mononuclear Cells

Freshly isolated human peripheral blood mononuclear cells were grown inthe presence of IL-29 (20 ng/mL), IFNα2a (2 ng/ml) (PBL Biomedical Labs,Piscataway, N.J.), or in medium alone. Cells were incubated for 6, 24,48, or 72 hours, and then total RNA was isolated and treated withRNase-free DNase. 100 ng total RNA was used as a template for One-StepSemi-Quantitative RT-PCR® using Taqman One-Step RT-PCR Master Mix®Reagents and gene specific primers as suggested by the manufacturer.(Applied Biosystems, Branchburg, N.J.) Results were normalized to HPRTand are shown as the fold induction over the medium alone control foreach time-point. Table 13 shows that IL-29 induces Interferon StimulatedGene Expression in human peripheral blood mononuclear cells at alltime-points tested. TABLE 13 MxA Fold Pkr Fold OAS Fold inductionInduction Induction  6 hr IL29 3.1 2.1 2.5  6 hr IFNα2a 17.2 9.6 16.2 24hr IL29 19.2 5.0 8.8 24 hr IFNα2a 57.2 9.4 22.3 48 hr IL29 7.9 3.5 3.348 hr IFNα2a 18.1 5.0 17.3 72 hr IL29 9.4 3.7 9.6 72 hr IFNα2a 29.9 6.447.3B. Activated Human T Cells

Human T cells were isolated by negative selection from freshly harvestedperipheral blood mononuclear cells using the Pan T-cell Isolation® kitaccording to manufacturer's instructions (Miltenyi, Auburn, Calif.). Tcells were then activated and expanded for 5 days with plate-boundanti-CD3, soluble anti-CD28 (0.5 ug/ml), (Pharmingen, San Diego, Calif.)and Interleukin 2 (IL-2; 100 U/ml) (R&D Systems, Minneapolis, Minn.),washed and then expanded for a further 5 days with IL-2. Followingactivation and expansion, cells were stimulated with IL-28A (20 ng/ml),IL-29 (20 ng/ml), or medium alone for 3, 6, or 18 hours. Total RNA wasisolated and treated with RNase-Free DNase. One-Step Semi-QuantitativeRE-PCR® was performed as described in the example above. Results werenormalized to HPRT and are shown as the fold induction over the mediumalone control for each time-point. Table 14 shows that IL-28 and IL-29induce Interferon Stimulated Gene expression in activated human T cellsat all time-points tested. TABLE 14 MxA Fold Pkr Fold OAS Fold InductionInduction Induction Donor #1 3 hr IL28 5.2 2.8 4.8 Donor #1 3 hr IL295.0 3.5 6.0 Donor #1 6 hr IL28 5.5 2.2 3.0 Donor #1 6 hr IL29 6.4 2.23.7 Donor #1 18 hr IL28 4.6 4.8 4.0 Donor #1 18 hr IL29 5.0 3.8 4.1Donor #2 3 hr IL28 5.7 2.2 3.5 Donor #2 3 hr IL29 6.2 2.8 4.7 Donor #2 6hr IL28 7.3 1.9 4.4 Donor #2 6 hr IL29 8.7 2.6 4.9 Donor #2 18 hr IL284.7 2.3 3.6 Donor #2 18 hr IL29 4.9 2.1 3.8C. Primary Human Hepatocytes

Freshly isolated human hepatocytes from two separate donors (Cambrex,Baltimore, Md. and CellzDirect, Tucson, Ariz.) were stimulated withIL-28A (50 ng/ml), IL-29 (50 ng/ml), IFNα2a (50 ng/ml), or medium alonefor 24 hours. Following stimulation, total RNA was isolated and treatedwith RNase-Free DNase. One-step semi-quantitative RE-PCR was performedas described previously in the example above. Results were normalized toHPRT and are shown as the fold induction over the medium alone controlfor each time-point. Table 15 shows that IL-28 and IL-29 induceInterferon Stimulated Gene expression in primary human hepatocytesfollowing 24-hour stimulation. TABLE 15 MxA Fold Pkr Fold OAS FoldInduction Induction Induction Donor #1 IL28 31.4 6.4 30.4 Donor #1 IL2931.8 5.2 27.8 Donor #1 IFN-α2a 63.4 8.2 66.7 Donor #2 IL28 41.7 4.2 24.3Donor #2 IL29 44.8 5.2 25.2 Donor #2 IFN-α2a 53.2 4.8 38.3D. HepG2 and HuH7: Human Liver Hepatoma Cell Lines

HepG2 and HuH7 cells (ATCC NOS. 8065, Manassas, Va were stimulated withIL-28A (10 ng/ml), IL-29 (10 ng/ml), IFNα2a (10 ng/ml), IFNB (1 ng/ml)(PBL Biomedical, Piscataway, N.J.), or medium alone for 24 or 48 hours.In a separate culture, HepG2 cells were stimulated as described abovewith 20 ng/ml of MetIL-29C172S-PEG or MetIL-29-PEG. Total RNA wasisolated and treated with RNase-Free DNase. 100 ng Total RNA was used asa template for one-step semi-quantitative RE-PCR as describedpreviously. Results were normalized to HPRT and are shown as the foldinduction over the medium alone control for each time-point. Table 16shows that IL-28 and IL-29 induce ISG expression in HepG2 and HuH7 liverhepatoma cell lines after 24 and 48 hours. TABLE 16 MxA Fold Pkr FoldOAS Fold Induction Induction Induction HepG2 24 hr IL28 12.4 0.7 3.3HepG2 24 hr IL29 36.6 2.2 6.4 HepG2 24 hr IFNα2a 12.2 1.9 3.2 HepG2 24hr IFNβ 93.6 3.9 19.0 HepG2 48 hr IL28 2.7 0.9 1.1 HepG2 48 hr IL29 27.22.1 5.3 HepG2 48 hr IFNα2a 2.5 0.9 1.2 HepG2 48 hr IFNβ 15.9 1.8 3.3HuH7 24 hr IL28 132.5 5.4 52.6 HuH7 24 hr IL29 220.2 7.0 116.6 HuH7 24hr IFNα2a 157.0 5.7 67.0 HuH7 24 hr IFNβ 279.8 5.6 151.8 HuH7 48 hr IL2825.6 3.4 10.3 HuH7 48 hr IL29 143.5 7.4 60.3 HuH7 48 hr IFNα2a 91.3 5.832.3 HuH7 48 hr IFNβ 65.0 4.2 35.7

TABLE 17 MxA Fold OAS Fold Pkr Fold Induction Induction InductionMetIL-29-PEG 36.7 6.9 2.2 MetIL-29C172S-PEG 46.1 8.9 2.8

Data shown is for 20 ng/ml metIL-29-PEG and metIL-29C172S-PEG versionsof IL-29 after culture for 24 hours.

Data shown is normalized to HPRT and shown as fold induction overunstimulated cells.

Example 14

Antiviral Activity of IL-28 and IL-29 in HCV Replicon System

The ability of antiviral drugs to inhibit HCV replication can be testedin vitro with the HCV replicon system. The replicon system consists ofthe Huh7 human hepatoma cell line that has been transfected withsubgenomic RNA replicons that direct constitutive replication of HCVgenomic RNAs (Blight, K. J. et al. Science 290:1972-1974, 2000).Treatment of replicon clones with IFNα at 10 IU/ml reduces the amount ofHCV RNA by 85% compared to untreated control cell lines. The ability ofIL-28A and IL-29 to reduce the amount of HCV RNA produced by thereplicon clones in 72 hours indicates the antiviral state conferred uponHuh7 cells by IL-28A/IL-29 treatment is effective in inhibiting HCVreplicon replication, and thereby, very likely effective in inhibitingHCV replication.

The ability of IL-28A and IL-29 to inhibit HCV replication as determinedby Bayer Branched chain DNA kit, is be done under the followingconditions:

IL28 alone at increasing concentrations (6)* up to 1.0 μg/ml

IL29 alone at increasing concentrations (6)* up to 1.0 ∞g/ml

PEGIL29 alone at increasing concentrations (6)* up to 1.0 μg/ml

IFNα2A alone at 0.3, 1.0, and 3.0 IU/ml

Ribavirin alone.

The positive control is IFNα and the negative control is ribavirin.

The cells are stained after 72 hours with Alomar Blue to assessviablility.

*The concentrations for conditions 1-3 are:

μg/ml, 0.32 μg/ml, 0.10 μg/ml, 0.032 μg/ml, 0.010 μg/ml, 0.0032 μg/ml.

The replicon clone (BB7) is treated 1× per day for 3 consecutive dayswith the doses listed above. Total HCV RNA is measured after 72 hours.

Example 15

IL-28 and IL-29 Have Antiviral Activity Against Pathogenic Viruses

Two methods are used to assay in vitro antiviral activity of IL-28 andIL-29 against a panel of pathogenic viruses including, among others,adenovirus, parainfluenza virus, respiratory syncytial virus, rhinovirus, coxsackie virus, influenza virus, vaccinia virus, west nilevirus, dengue virus, venezuelan equine encephalitis virus, pichindevirus and polio virus. These two methods are inhibition of virus-inducedcytopathic effect (CPE) determined by visual (microscopic) examinationof the cells and increase in neutral red (NR) dye uptake into cells. Inthe CPE inhibition method, seven concentrations of test drug (log10dilutions, such as 1000, 100, 10, 1, 0.1, 0.01, 0.001 ng/ml) areevaluated against each virus in 96-well flat-bottomed microplatescontaining host cells. The compounds are added 24 hours prior to virus,which is used at a concentration of approximately 5 to 100 cell cultureinfectious doses per well, depending upon the virus, which equates to amultiplicity of infection (MOI) of 0.01 to 0.0001 infectious particlesper cell. The tests are read after incubation at 37° C. for a specifiedtime sufficient to allow adequate viral cytopathic effect to develop. Inthe NR uptake assay, dye (0.34% concentration in medium) is added to thesame set of plates used to obtain the visual scores. After 2 h, thecolor intensity of the dye absorbed by and subsequently eluted from thecells is determined using a microplate autoreader. Antiviral activity isexpressed as the 50% effective (virus-inhibitory) concentration (EC50)determined by plotting compound concentration versus percent inhibitionon semilogarithmic graph paper. The EC50/IC50 data in some cases may bedetermined by appropriate regression analysis software. In general, theEC50s determined by NR assay are two-to fourfold higher than thoseobtained by the CPE method. TABLE 18 Visual Assay SI Visual (IC50/ VirusCell line Drug EC50 Visual IC50 Visual EC50) Adenovirus A549 IL-28A >10μg/ml >10 μg/ml 0 Adenovirus A549 IL-29 >10 μg/ml >10 μg/ml 0 AdenovirusA549 MetIL-29C172S- >10 μg/ml >10 μg/ml 0 PEG Parainfluenza MA-104IL-28A >10 μg/ml >10 μg/ml 0 virus Parainfluenza MA-104 IL-29 >10μg/ml >10 μg/ml 0 virus Parainfluenza MA-104 MetIL-29C172S- >10μg/ml >10 μg/ml 0 virus PEG Respiratory MA-104 IL-28A >10 μg/ml >10μg/ml 0 syncytial virus Respiratory MA-104 IL-29 >10 μg/ml >10 μg/ml 0syncytial virus Respiratory MA-104 MetIL-29C172S- >10 μg/ml >10 μg/ml 0syncytial PEG virus Rhino 2 KB IL-28A >10 μg/ml >10 μg/ml 0 Rhino 2 KBIL-29 >10 μg/ml >10 μg/ml 0 Rhino 2 KB MetIL-29C172S- >10 μg/ml >10μg/ml 0 PEG Rhino 9 HeLa IL-28A >10 μg/ml >10 μg/ml 0 Rhino 9 HeLaIL-29 >10 μg/ml >10 μg/ml 0 Rhino 9 HeLa MetIL-29C172S- >10 μg/ml >10μg/ml 0 PEG Coxsackie KB IL-28A >10 μg/ml >10 μg/ml 0 B4 virus CoxsackieKB IL-29 >10 μg/ml >10 μg/ml 0 B4 virus Coxsackie KB MetIL-29C172S- >10μg/ml >10 μg/ml 0 B4 virus PEG Influenza Maden- IL-28A >10 μg/ml >10μg/ml 0 (type A Darby [H3N2]) Canine Kidney Influenza Maden- IL-29 >10μg/ml >10 μg/ml 0 (type A Darby [H3N2]) Canine Kidney Influenza Maden-MetIL-29C172S- >10 μg/ml >10 μg/ml 0 (type A Darby PEG [H3N2]) CanineKidney Influenza Vero IL-28A   0.1 μg/ml >10 μg/ml >100 (type A [H3N2])Influenza Vero IL-29 >10 μg/ml >10 μg/ml 0 (type A [H3N2]) InfluenzaVero MetIL-29C172S- 0.045 μg/ml  >10 μg/ml >222 (type A PEG [H3N2])Vaccinia Vero IL-28A >10 μg/ml >10 μg/ml 0 virus Vaccinia Vero IL-29 >10μg/ml >10 μg/ml 0 virus Vaccinia Vero MetIL-29C172S- >10 μg/ml >10 μg/ml0 virus PEG West Nile Vero IL-28A 0.00001 μg/ml   >10 μg/ml >1,000,000virus West Nile Vero IL-29 0.000032 μg/ml   >10 μg/ml >300,000 virusWest Nile Vero MetIL-29C172S- 0.001 μg/ml  >10 μg/ml >10,000 virus PEGDengue virus Vero IL-28A 0.01 μg/ml >10 μg/ml >1000 Dengue virus VeroIL-29 0.032 μg/ml  >10 μg/ml >312 Dengue virus Vero MetIL-29C172S-0.0075 μg/ml  >10 μg/ml >1330 PEG Venezuelan Vero IL-28A 0.01 μg/ml >10μg/ml >1000 equine encephalitis virus Venezuelan Vero IL-29 0.012μg/ml  >10 μg/ml >833 equine encephalitis virus Venezuelan VeroMetIL-29C172S- 0.0065 μg/ml  >10 μg/ml >1538 equine PEG encephalitisvirus Pichinde BSC-1 IL-28A >10 μg/ml >10 μg/ml 0 virus Pichinde BSC-1IL-29 >10 μg/ml >10 μg/ml 0 virus Pichinde BSC-1 MetIL-29C172S- >10μg/ml >10 μg/ml 0 virus PEG Polio virus Vero IL-28A >10 μg/ml >10 μg/ml0 Polio virus Vero IL-29 >10 μg/ml >10 μg/ml 0 Polio virus VeroMetIL-29C172S- >10 μg/ml >10 μg/ml 0 PEG

TABLE 19 Neutral Red Assay SI NR (IC50/ Virus Cell line Drug EC50 NRIC50 NR EC50) Adenovirus A549 IL-28A >10 μg/ml >10 μg/ml 0 AdenovirusA549 IL-29 >10 μg/ml >10 μg/ml 0 Adenovirus A549 MetIL-29C172S- >10μg/ml >10 μg/ml 0 PEG Parainfluenza MA-104 IL-28A >10 μg/ml >10 μg/ml 0virus Parainfluenza MA-104 IL-29 >10 μg/ml >10 μg/ml 0 virusParainfluenza MA-104 MetIL-29C172S- >10 μg/ml >10 μg/ml 0 virus PEGRespiratory MA-104 IL-28A >10 μg/ml >10 μg/ml 0 syncytial virusRespiratory MA-104 IL-29 >10 μg/ml >10 μg/ml 0 syncytial virusRespiratory MA-104 MetIL-29C172S- 5.47 μg/ml >10 μg/ml >2 syncytialvirus PEG Rhino 2 KB IL-28A >10 μg/ml >10 μg/ml 0 Rhino 2 KB IL-29 >10μg/ml >10 μg/ml 0 Rhino 2 KB MetIL-29C172S- >10 μg/ml >10 μg/ml 0 PEGRhino 9 HeLa IL-28A 1.726 μg/ml  >10 μg/ml >6 Rhino 9 HeLa IL-29 0.982μg/ml  >10 μg/ml >10 Rhino 9 HeLa MetIL-29C172S- 2.051 μg/ml  >10μg/ml >5 PEG Coxsackie B4 KB IL-28A >10 μg/ml >10 μg/ml 0 virusCoxsackie B4 KB IL-29 >10 μg/ml >10 μg/ml 0 virus Coxsackie B4 KBMetIL-29C172S- >10 μg/ml >10 μg/ml 0 virus PEG Influenza (type Maden-IL-28A >10 μg/ml >10 μg/ml 0 A [H3N2]) Darby Canine Kidney Influenza(type Maden- IL-29 >10 μg/ml >10 μg/ml 0 A [H3N2]) Darby Canine KidneyInfluenza (type Maden- MetIL-29C172S- >10 μg/ml >10 μg/ml 0 A [H3N2])Darby PEG Canine Kidney Influenza (type Vero IL-28A 0.25 μg/ml >10μg/ml >40 A [H3N2]) Influenza (type Vero IL-29    2 μg/ml >10 μg/ml >5 A[H3N2]) Influenza (type Vero MetIL-29C172S-  1.4 μg/ml >10 μg/ml >7 A[H3N2]) PEG Vaccinia virus Vero IL-28A >10 μg/ml >10 μg/ml 0 Vacciniavirus Vero IL-29 >10 μg/ml >10 μg/ml 0 Vaccinia virus VeroMetIL-29C172S- >10 μg/ml >10 μg/ml 0 PEG West Nile virus Vero IL-28A0.0001 μg/ml  >10 μg/ml >100,000 West Nile virus Vero IL-29 0.00025μg/ml   >10 μg/ml >40,000 West Nile virus Vero MetIL-29C172S- 0.00037μg/ml   >10 μg/ml >27,000 PEG Dengue virus Vero IL-28A  0.1 μg/ml >10μg/ml >100 Dengue virus Vero IL-29 0.05 μg/ml >10 μg/ml >200 Denguevirus Vero MetIL-29C172S- 0.06 μg/ml >10 μg/ml >166 PEG Venezuelan VeroIL-28A 0.035 μg/ml  >10 μg/ml >286 equine encephalitis virus VenezuelanVero IL-29 0.05 μg/ml >10 μg/ml >200 equine encephalitis virusVenezuelan Vero MetIL-29C172S- 0.02 μg/ml >10 μg/ml >500 equine PEGencephalitis virus Pichinde virus BSC-1 IL-28A >10 μg/ml >10 μg/ml 0Pichinde virus BSC-1 IL-29 >10 μg/ml >10 μg/ml 0 Pichinde virus BSC-1MetIL-29C172S- >10 μg/ml >10 μg/ml 0 PEG Polio virus Vero IL-28A >1.672μg/ml   >10 μg/ml >6 Polio virus Vero IL-29 >10 μg/ml >10 μg/ml 0 Poliovirus Vero MetIL-29C172S- >10 μg/ml >10 μg/ml 0 PEG

Example 16

IL-28 IL-29 metIL-29-PEG and metIL-29C172S-PEG Stimulate ISG Inductionin the Mouse Liver Cell Line AML-12

Interferon stimulated genes (ISGs) are genes that are induced by type Iinterferons (IFNs) and also by the IL-28 and IL-29 family molecules,suggesting that IFN and IL-28 and IL-29 induce similar pathways leadingto antiviral activity. Human type I IFNs (IFNα1-4 and IFNβ) have littleor no activity on mouse cells, which is thought to be caused by lack ofspecies cross-reactivity. To test if human IL-28 and IL-29 have effectson mouse cells, ISG induction by human IL-28 and IL-29 was evaluated byreal-time PCR on the mouse liver derived cell line AML-12.

AML-12 cells were plated in 6-well plates in complete DMEM media at aconcentration of 2×10⁶ cells/well. Twenty-four hours after platingcells, human IL-28 and IL-29 were added to the culture at aconcentration of 20 ng/ml. As a control, cells were either stimulatedwith mouse IFNα (positive control) or unstimulated (negative). Cellswere harvested at 8, 24, 48 and 72 hours after addition of CHO-derivedhuman IL-28A (SEQ ID NO:18) or IL-29 (SEQ ID NO:20). RNA was isolatedfrom cell pellets using RNAEasy-kit® (Qiagen, Valencia, Calif.). RNA wastreated with DNase (Millipore, Billerica, Mass.) to clean RNA of anycontaminating DNA. cDNA was generated using Perkin-Elmer RE mix. ISGgene induction was evaluated by real-time PCR using primers and probesspecific for mouse OAS, Pkr and Mx1. To obtain quantitative data, HPRTreal-time PCR was duplexed with ISG PCR. A standard curve was obtainedusing known amounts of RNA from IFN-stimulated mouse PBLs. All data areshown as expression relative to internal HPRT expression.

Human IL-28A and IL-29 stimulated ISG induction in the mouse hepatocytecell line AML-12 and demonstrated that unlike type I IFNs, the IL-28/29family proteins showed cross-species reactivity. TABLE 20 StimulationOAS PkR Mx1 None 0.001 0.001 0.001 Human IL-28 0.04 0.02 0.06 HumanIL-29 0.04 0.02 0.07 Mouse IL-28 0.04 0.02 0.08 Mouse IFNα 0.02 0.020.01

All data shown were expressed as fold relative to HPRT gene expressionng of OAS mRNA=normalized value of OAS mRNA amount relative to internal.

ng of HPRT mRNA housekeeping gene, HPRT.

As an example, the data for the 48 hour time point is shown. TABLE 21AML12's Mx1 Fold OAS Fold Pkr Fold Induction Induction InductionMetIL-29-PEG 728 614 8 MetIL-29C172S-PEG 761 657 8

Cells were stimulated with 20 ng/ml metIL-29-PEG or metIL-29C172S-PEGfor 24 hours.

Data shown is normalized to HPRT and shown as fold induction overunstimulated cells.

Example 17

ISGs are Efficiently Induced in Spleens of Transgenic Mice ExpressingHuman IL-29

Transgenic (Tg) mice were generated expressing human IL-29 under thecontrol of the Eu-lck promoter. To study if human IL-29 has biologicalactivity in vivo in mice, expression of ISGs was analyzed by real-timePCR in the spleens of Eu-lck IL-29 transgenic mice.

Transgenic mice (C3H/C57BL/6) were generated using a construct thatexpressed the human IL-29 gene under the control of the Eu-lck promoter.This promoter is active in T cells and B cells. Transgenic mice andtheir non-transgenic littermates (n=2/gp) were sacrificed at about 10weeks of age. Spleens of mice were isolated. RNA was isolated from cellpellets using RNAEasy-kit® (Qiagen). RNA was treated with DNase to cleanRNA of any contaminating DNA. cDNA was generated using Perkin-Elmer RE®mix. ISG gene induction was evaluated by real-time PCR using primers andprobes (5′FAM, 3′NFQ) specific for mouse OAS, Pkr and Mx1. To obtainquantitative data, HPRT real-time PCR was duplexed with ISG PCR.Furthermore, a standard curve was obtained using known amounts of IFNstimulated mouse PBLs. All data are shown as expression relative tointernal HPRT expression.

Spleens isolated from IL-29 Tg mice showed high induction of ISGs OAS,Pkr and Mx1 compared to their non-Tg littermate controls suggesting thathuman IL-29 is biologically active in vivo in mice. TABLE 22 Mice OASPkR Mx1 Non-Tg 4.5 4.5 3.5 IL-29 Tg 12 8 21

All data shown are fold expression relative to HPRT gene expresssion.The average expression in two mice is shown.

Example 18

Human IL-28 and IL-29 Protein Induce ISG Gene Expression In Liver,Spleen and Blood of Mice

To determine whether human IL-28 and IL-29 induce interferon stimulatedgenes in vivo, CHO-derived human IL-28A and IL-29 protein were injectedinto mice. In addition, E. coli derived IL-29 was also tested in vivoassays as described above using MetIL-29C172S-PEG and MetIL-29-PEG. Atvarious time points and at different doses, ISG gene induction wasmeasured in the blood, spleen and livers of the mice.

C57BL/6 mice were injected i.p or i.v with a range of doses (10 μg -250μg) of CHO-derived human IL-28A and IL-29 or MetIL-29C172S-PEG andMetIL-29C16-C113-PEG. Mice were sacrificed at various time points (1hr-48 hr). Spleens and livers were isolated from mice, and RNA wasisolated. RNA was also isolated from the blood cells. The cells werepelleted and RNA isolated from pellets using RNAEasy®-kit (Qiagen). RNAwas treated with DNase (Amicon) to rid RNA of any contaminating DNA.cDNA was generated using Perkin-Elmer RE mix (Perkin-Elmer). ISG geneinduction was measured by real-time PCR using primers and probesspecific for mouse OAS, Pkr and Mx1. To obtain quantitative data, HPRTreal-time PCR was duplexed with ISG PCR. A standard curve was calculatedusing known amounts of IFN-stimulated mouse PBLs. All data are shown asexpression relative to internal HPRT expression.

Human IL-29 induced ISG gene expression (OAS, Pkr, Mx1) in the livers,spleen and blood of mice in a dose dependent manner. Expression of ISGspeaked between 1-6 hours after injection and showed sustained expressionabove control mice up to 48 hours. In this experiment, human IL-28A didnot induce ISG gene expression. TABLE 23 Injection OAS-1 hr OAS-6 hrOAS-24 hr OAS-48 hr None - liver 1.6 1.6 1.6 1.6 IL-29 liver 2.5 4 2.52.8 None - spleen 1.8 1.8 1.8 1.8 IL-29 - spleen 4 6 3.2 3.2 None -blood 5 5 5 5 IL-29 blood 12 18 11 10

Results shown are fold expression relative to HPRT gene expression. Asample data set for IL-29 induced OAS in liver at a single injection of250 μg i.v. is shown. The data shown is the average expression from 5different animals/group. TABLE 24 Injection OAS (24 hr) None 1.8 IL-2910 μg 3.7 IL-29 50 μg 4.2 IL-29 250 μg 6

TABLE 25 MetIL-29-PEG MetIL-29C172S-PEG Naive 3 hr 6 hr 12 hr 24 hr 3 hr6 hr 12 hr 24 hr 24 hr PKR 18.24 13.93 4.99 3.77 5.29 5.65 3.79 3.553.70 OAS 91.29 65.93 54.04 20.81 13.42 13.02 10.54 8.72 6.60 Mx1 537.51124.99 33.58 35.82 27.89 29.34 16.61 0.00 10.98

Mice were injected with 100 μg of proteins i.v. Data shown is foldexpression over HPRT expression from livers of mice. Similar data wasobtained from blood and spleens of mice.

Example 19

IL-28 and IL-29 Induce ISG Protein In Mice

To analyze of the effect of human IL-28 and IL-29 on induction of ISGprotein (OAS), serum and plasma from IL-28 and IL-29 treated mice weretested for OAS activity.

C57BL/6 mice were injected i.v with PBS or a range of concentrations (10μg-250 μg) of human IL-28 or IL-29. Serum and plasma were isolated frommice at varying time points, and OAS activity was measured using the OASradioimmunoassay (RIA) kit from Eiken Chemicals (Tokyo, Japan).

IL-28 and IL-29 induced OAS activity in the serum and plasma of miceshowing that these proteins are biologically active in vivo. TABLE 26Injection OAS-1 hr OAS-6 hr OAS-24 hr OAS-48 hr None 80 80 80 80 IL-2980 80 180 200

OAS activity is shown at pmol/dL of plasma for a single concentration(250 μg) of human IL-29.

Example 20

IL-28 and IL-29 Inhibit Adenoviral Pathology in Mice

To test the antiviral activities of IL-28 and IL-29 against viruses thatinfect the liver, the test samples were tested in mice againstinfectious adenoviral vectors expressing an internal green fluorescentprotein (GFP) gene. When injected intravenously, these viruses primarilytarget the liver for gene expression. The adenoviruses are replicationdeficient, but cause liver damage due to inflammatory cell infiltratethat can be monitored by measurement of serum levels of liver enzymeslike AST and ALT, or by direct examination of liver pathology.

C57B1/6 mice were given once daily intraperitoneal injections of 50 μgmouse IL-28 (zcyto24 as shown in SEQ ID NO:9) or metIL-29C172S-PEG for 3days. Control animals were injected with PBS. One hour following the3^(rd) dose, mice were given a single bolus intravenous tail veininjection of the adenoviral vector, AdGFP (1×10⁹ plaque-forming units(pfu)). Following this, every other day mice were given an additionaldose of PBS, mouse IL-28 or metIL-29C172S-PEG for 4 more doses (total of7 doses). One hour following the final dose of PBS, mouse IL-28 ormetIL-29C172S-PEG mice were terminally bleed and sacrificed. The serumand liver tissue were analyzed. Serum was analyzed for AST and ALT liverenzymes. Liver was isolated and analyzed for GFP expression andhistology. For histology, liver specimens were fixed in formalin andthen embedded in paraffin followed by H&E staining. Sections of liverthat had been blinded to treat were examined with a light microscope.Changes were noted and scored on a scale designed to measure liverpathology and inflammation.

Mouse IL-28 and IL-29 inhibited adenoviral infection and gene expressionas measured by liver fluorescence. PBS-treated mice (n=8) had an averagerelative liver fluorescence of 52.4 (arbitrary units). In contrast,IL-28-treated mice (n=8) had a relative liver fluorescence of 34.5, andIL-29-treated mice (n=8) had a relative liver fluorescence of 38.9. Areduction in adenoviral infection and gene expression led to a reducedliver pathology as measured by serum ALT and AST levels and histology.PBS-treated mice (n=8) had an average serum AST of 234 U/L (units/liter)and serum ALT of 250 U/L. In contrast, IL-28-treated mice (n=8) had anaverage serum AST of 193 U/L and serum ALT of 216 U/L, and IL-29-treatedmice (n=8) had an average serum AST of 162 U/L and serum ALT of 184 U/L.In addition, the liver histology indicated that mice given either mouseIL-28 or IL-29 had lower liver and inflammation scores than thePBS-treated group. The livers from the IL-29 group also had lessproliferation of sinusoidal cells, fewer mitotic figures and fewerchanges in the hepatocytes (e.g. vacuolation, presence of multiplenuclei, hepatocyte enlargement) than in the PBS treatment group. Thesedata demonstrate that mouse IL-28 and IL-29 have antiviral propertiesagainst a liver-trophic virus.

Example 21

LCMV Models

Lymphocytic choriomeningitis virus (LCMV) infections in mice are anexcellent model of acture and chronic infection. These models are usedto evaluate the effect of cytokines on the antiviral immune response andthe effects IL-28 and IL-29 have viral load and the antiviral immuneresponse. The two models used are: LCMV Armstrong (acute) infection andLCMV Clone 13 (chronic) infection. (See, e.g., Wherry et al., J. Virol.77:4911-4927, 2003; Blattman et al., Nature Med. 9(5):540-547, 2003;Hoffinan et al., J. Immunol. 170:1339-1353, 2003.) There are threestages of CD8 T cell development in response to virus:1) expansion, 2)contraction, and 3) memory (acute model). IL-28 or IL-29 is injectedduring each stage for both acute and chronic models. In the chronicmodel, IL-28 or IL-29 is injected 60 days after infection to assess theeffect of IL-28 or IL-29 on persistent viral load. For both acute andchronic models, IL-28 or IL-29 is injected, and the viral load in blood,spleen and liver is examined. Other paramenter that can be examinedinclude: tetramer staining by flow to count the number of LCMV-specificCD8+ T cells; the ability of tetramer+ cells to produce cytokines whenstimulated with their cognate LCMV antigen; and the ability ofLCMV-specific CD8+ T cells to proliferate in response to their cognateLCMV antigen. LCMV-specific T cells are phenotyped by flow cytometry toassess the cells activation and differentiation state. Also, the abilityof LCMV-specific CTL to lyse target cells bearing their cognate LCMVantigen is examined. The number and function of LCMV-specific CD4+ Tcells is also assessed.

A reduction in viral load after treatment with IL-28 or IL-29 isdetermined. A 50% reduction in viral load in any organ, especiallyliver, would be significant. For IL-28 or IL-29 treated mice, a 20%increase in the percentage of tetramer positive T cells thatproliferate, make cytokine, or display a mature phenotype relative tountreated mice would also be considered significant.

IL-28 or IL-29 injection leading to a reduction in viral load is due tomore effective control of viral infection especially in the chronicmodel where untreated the viral titers remain elevated for an extendedperiod of time. A two fold reduction in viral titer relative tountreated mice is considered significant.

Example 22

Influenza Model of Acute Viral Infection

A. Preliminary Experiment to Test Antiviral Activity.

To determine the antiviral activity of IL-28 or IL-29 on acute infectionby Influenza virus, an in vivo study using influenza infected c57B1/6mice is performed using the following protocol:

Animals:6 weeks-old female BALB/c mice (Charles River) with 148 mice, 30per group.

Groups:

Absolute control (not infected) to run in parallel for antibody titreand histopathology (2 animals per group).

Vehicle (i.p.) saline.

Amantadine (positive control) 10 mg/day during 5 days (per os) starting2 hours before infection.

IL-28 or IL-29 treated (5 μg, i.p. starting 2 hours after infection).

IL-28 or IL-29 (25 μg, i.p. starting 2 hours after infection).

IL-28 or IL-29 (125 μg, i.p. starting 2 hours after infection).

Day 0—Except for the absolute controls, all animals infected withInfluenza virus.

For viral load (10 at LD50).

For immunology workout (LD30).

Day 0-9—daily injections of IL-28 or IL-29 (i.p.).

Body weight and general appearance recorded (3 times/week).

Day 3—sacrifice of 8 animals per group

Viral load in right lung (TCID50).

Histopathology in left lung.

Blood sample for antibody titration.

Day 10—sacrifice of all surviving animals collecting blood samples forantibody titration, isolating lung lymphocytes (4 pools of 3) for directCTL assay (in all 5 groups), and quantitative immunophenotyping for thefollowing markers: CD3/CD4, CD3/CD8, CD3/CD8/CD11b, CD8/CD44/CD62L,CD3/DX5, GR-1/F480, and CD19.

Study No.2

Efficacy study of IL-28 or IL-29 in C57B1/6 mice infected withmouse-adapted virus is done using 8 weeks-old female C57B1/6 mice(Charles River).

Group 1: Vehicle (i.p.).

Group 2: Positive control: Anti-influenza neutralizing antibody (goatanti-influenza A/USSR (H1N1) (Chemicon International, Temecula, Calif.);40 μg/mouse at 2 h and 4 h post infection (10 μl intranasal).

Group 3:IL-28 or IL-29 (5 μg, i.p.).

Group 4:IL-28 or IL-29 (25 μg, i.p.).

Group 5:IL-28 or IL-29 (125 μg, i.p.).

Following-life observations and immunological workouts are prepared:

Day 0—all animals infected with Influenza virus (dose determined inexperiment 2).

Day 0-9—daily injections of IL-28 or IL-29 (i.p.).

Body weight and general appearance recorded every other day.

Day 10—sacrifice of surviving animals and perform viral assay todetermine viral load in lung.

Isolation of lung lymphocytes (for direct CTL assay in the lungs usingEL-4 as targets and different E:T ratio (based on best results fromexperiments 1 and 2).

Tetramer staining: The number of CD8+ T cells binding MHC Class Itetramers containing influenza A nucleoprotein (NP) epitope are assessedusing complexes of MHC class I with viral peptides: FLU-NP₃₆₆-₃₇₄/D^(b)(ASNENME™), (LMCV peptide/D^(b)).

Quantitative immunophenotyping of the following: CD8, tetramer,intracellular IFNγ, NK1.1, CD8, tetramer, CD62L, CD44, CD3(+ or −),NK1.1(+), intracellular IFNγ, CD4, CD8, NK1.1, DX5, CD3 (+ or −), NK1.1,DX5, tetramer, Single colour samples for cytometer adjustment.

Survival Re-Challenge Study

Day 30:Survival study with mice are treated for 9 days with differentdoses of IL-28 or IL-29 or with positive anti-influenza antibodycontrol. Body weight and antibody production in individual serum samples(Total, IgGI, IgG2a, IgG2b) are measured.

Re-Challenge Study:

Day 0:Both groups will be infected with A/PR virus (1LD30).

Group 6 will not be treated.

Group 7 will be treated for 9 days with 125 μg of IL-28 or IL-29.

Day 30:Survival study

Body weight and antibody production in individual serum samples (Total,IgG1, IgG2a, IgG2b) are measured.

Day 60:Re-challenge study.

Survivors in each group will be divided into 2 subgroups.

Group 6A and 7A will be re-challenge with A/PR virus (1 LD30).

Group 6B and 7B will be re-challenge with A/PR virus (1 LD30).

Both groups will be followed up and day of sacrifice will be determined.Body weight and antibody production in individual serum samples (Total,IgG1, IgG2a, IgG2b) are measured.

Example 21

IL-28 and IL-29 Have Antiviral Activity Against Hepatitis B Virus (HBV)In Vivo.

A transgenic mouse model (Guidoffi et al., J. Virology 69:6158-6169,1995) supports the replication of high levels of infectious HBV and hasbeen used as a chemotherapeutic model for HBV infection. Transgenic miceare treated with antiviral drugs and the levels of HBV DNA and RNA aremeasured in the transgenic mouse liver and serum following treatment.HBV protein levels can also be measured in the transgenic mouse serumfollowing treatment. This model has been used to evaluate theeffectiveness of lamivudine and IFN-α in reducing HBV viral titers.

HBV TG mice (male) are given intraperitoneal injections of 2.5, 25 or250 micrograms IL-28 or IL-29 every other day for 14 days (total of 8doses). Mice are bled for serum collection on day of treatment (day 0)and day 7. One hour following the final dose of IL-29 mice undergo aterminal bleed and are sacrificed. Serum and liver are analyzed forliver HBV DNA, liver HBV RNA, serum HBV DNA, liver HBc, serum Hbe andserum HBs.

Reduction in liver HBV DNA, liver HBV RNA, serum HBV DNA, liver HBc,serum Hbe or serum HBs in response to IL-28 or IL-29 reflects antiviralactivity of these compounds against HBV.

Example 22

IL-28 and IL-29 Inhibit Human Herpesvirus-8 (HHV-8) replication inBCBL-1 Cells

The antiviral activities of IL-28 and IL-29 were tested against HHV-8 inan in vitro infection system using a B-lymphoid cell line, BCBL-1.

In the HHV-8 assay the test compound and a ganciclovir control wereassayed at five concentrations each, diluted in a half-log series. Theendpoints were TaqMan PCR for extracellular HHV-8 DNA (IC50) and cellnumbers using CellTiter96® reagent (TC50; Promega, Madison, Wis.).Briefly, BCBL-1 cells were plated in 96-well microtiter plates. After16-24 hours the cells were washed and the medium was replaced withcomplete medium containing various concentrations of the test compoundin triplicate. Ganciclovir was the positive control, while media alonewas a negative control (virus control, VC). Three days later the culturemedium was replaced with fresh medium containing the appropriatelydiluted test compound. Six days following the initial administration ofthe test compound, the cell culture supernatant was collected, treatedwith pronase and DNAse and then used in a real-time quantitative TaqManPCR assay. The PCR-amplified HHV-8 DNA was detected in real-time bymonitoring increases in fluorescence signals that result from theexonucleolytic degradation of a quenched fluorescent probe molecule thathybridizes to the amplified HHV-8 DNA. For each PCR amplification, astandard curve was simultaneously generated using dilutions of purifiedHHV-8 DNA. Antiviral activity was calculated from the reduction in HHV-8DNA levels (IC₅₀). A novel dye uptake assay was then employed to measurecell viability which was used to calculate toxicity (TC₅₀). Thetherapeutic index (TI) is calculated as TC₅₀1/IC₅₀.

IL-28 and IL-29 inhibit HHV-8 viral replication in BCBL-1 cells. IL-28Ahad an IC₅₀ of 1 μg/ml and a TC₅₀ of >10μg/ml (T1>10). IL-29 had an IC₅₀of 6.5 μg/ml and a TC₅₀ of >10 μg/ml (TI>1.85). MetIL-29C172S-PEG had anIC₅₀ of 0.14 μg/ml and a TC₅₀ of >10 μg/ml (TI>100).

Example 23

IL-28 and IL-29 Antiviral Activity Against Epstein Barr Virus (EBV)

The antiviral activities of IL-28 and IL-29 are tested against EBV in anin vitro infection system in a B-lymphoid cell line, P3HR-1. In the EBVassay the test compound and a control are assayed at five concentrationseach, diluted in a half-log series. The endpoints are TaqMan PCR forextracellular EBV DNA (IC50) and cell numbers using CellTiter96® reagent(TC50; Promega). Briefly, P3HR-1 cells are plated in 96-well microtiterplates. After 16-24 hours the cells are washed and the medium isreplaced with complete medium containing various concentrations of thetest compound in triplicate. In addition to a positive control, mediaalone is added to cells as a negative control (virus control, VC). Threedays later the culture medium is replaced with fresh medium containingthe appropriately diluted test compound. Six days following the initialadministration of the test compound, the cell culture supernatant iscollected, treated with pronase and DNAse and then used in a real-timequantitative TaqMan PCR assay. The PCR-amplified EBV DNA is detected inreal-time by monitoring increases in fluorescence signals that resultfrom the exonucleolytic degradation of a quenched fluorescent probemolecule that hybridizes to the amplified EBV DNA. For each PCRamplification, a standard curve was simultaneously generated usingdilutions of purified EBV DNA. Antiviral activity is calculated from thereduction in EBV DNA levels (IC₅₀). A novel dye uptake assay was thenemployed to measure cell viability which was used to calculate toxicity(TC₅₀). The therapeutic index (TI) is calculated as TC₅₀1/IC₅₀.

Example 24

IL-28 and IL-29 Antiviral Activity Against Herpes Simplex Virus-2(HSV-2)

The antiviral activities of IL-28 and IL-29 were tested against HSV-2 inan in vitro infection system in Vero cells. The antiviral effects ofIL-28 and IL-29 were assessed in inhibition of cytopathic effect assays(CPE). The assay involves the killing of Vero cells by the cytopathicHSV-2 virus and the inhibition of cell killing by IL-28 and IL-29. TheVero cells are propagated in Dulbecco's modified essential medium (DMEM)containing phenol red with 10% horse serum, 1% glutamine and 1%penicillin-streptomycin, while the CPE inhibition assays are performedin DMEM without phenol red with 2% FBS, 1% glutamine and 1% Pen-Strep.On the day preceding the assays, cells were trypsinized (1%trypsin-EDTA), washed, counted and plated out at 10⁴ cells/well in a96-well flat-bottom BioCoatiR plates (Fisher Scientific, Pittsburgh,Pa.) in a volume of 100 μl/well. The next morning, the medium wasremoved and a pre-titered aliquot of virus was added to the cells. Theamount of virus used is the maximum dilution that would yield completecell killing (>80%) at the time of maximal CPE development. Cellviability is determined using a CellTiter 96® reagent (Promega)according to the manufacturer's protocol, using a Vmax plate reader(Molecular Devices, Sunnyvale, Calif.). Compounds are tested at sixconcentrations each, diluted in assay medium in a half-log series.Acyclovir was used as a positive control. Compounds are added at thetime of viral infection. The average background and drug color-correcteddata for percent CPE reduction and percent cell viability at eachconcentration are determined relative to controls and the IC₅₀calculated relative to the TC₅₀.

IL-28A, IL-29 and MetIL-29C172S-PEG did not inhibit cell death (IC₅₀of >10 ug/ml) in this assay. There was also no antiviral activity ofIFNα in the is assay.

From the foregoing, it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention. Accordingly, the invention is notlimited except as by the appended claims.

1. A method of treating a viral infection comprising administering to animmunocompromised mammal with a hepatitis infection a therapeuticallyeffective amount of a polypeptide comprising the amino acid sequence ofSEQ ID NO:20, wherein after administration of the polypeptide thehepatitis infection is reduced.
 2. The method of claim 1 wherein thepolypeptide is conjugated to a polyalkyl oxide moiety.
 3. The method ofclaim 2 wherein the polyalkyl oxide moiety is a polyethylene glycol. 4.The method of claim 1 wherein the hepatitis infection is a hepatitis Binfection.
 5. The method of claim 1 wherein the hepatitis infection is ahepatitis C infection.